Syndecan enhancer element and syndecan stimulation of cellular differentiation

ABSTRACT

Methods are provided for altering levels of syndecan within a cell. The methods include enhancing syndecan expression via administration of growth factors, preventing suppression of syndecan expression via administration of anti-steroid agents, and altering syndecan biochemistry within the cell. The methods are used to induce or maintain cellular differentiation, and to decrease the growth of malignant cells. Application of the methods to the treatment of patients, including humans, is provided. A syndecan enhancer element, novel proteins that activate the enhancer element, non-human transgenic animals comprising this enhancer element linked to a structural gene, and the use of this enhancer element to regulate the expression of syndecan and other genes are also provided. The enhancer element can also be used to target expression of a gene to wound sites.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Application No. 08/206,186, filed Mar. 7, 1994 (abandoned), which is a continuation-in-part of PCT/FI93/00514, filed Dec. 1, 1993.

FIELD OF THE INVENTION

This invention is in the field of cancer biology and therapy. Specifically, the invention is directed to methods for altering the differentiated state of a cell by altering syndecan-1 expression. The method allows for the normalization of the growth rate and differentiation state of malignant cells, and is based on the stimulation of syndecan-1 expression in the malignant cells. Re-expression of syndecan-1 in such malignant cells promotes their normal differentiated phenotype and prevents their formation into tumors. This method may also be applied to normal cells to maintain their expression of genes characteristic of the differentiated state, e.g., the method may be used to prevent baldness by maintaining keratin production.

In addition, the invention is directed to transcription regulatory elements associated with the syndecan-1 gene, such as the FGF-inducible Response Element (FiRE) and to the use of these transcription regulatory elements for controlling gene expression. The FiRE can also be used to target expression of a reporter gene to wound sites in vivo.

BACKGROUND OF THE INVENTION

Cell surface proteoglycans play an important role in the regulation of cell behavior (Ruoslahti et al., Cell 64:867-869 (1991)). Through their covalently bound glycosaminoglycan side chains, such proteoglycans can bind various extracellular effector molecules (Jalkanen, et al., in Receptors for Extracellular Matrix, J. MacDonald & R. Mecham, eds., Academic Press, San Diego, pp. 1-37 (1991)). One central challenge in proteoglycan biology is to understand the biological consequences which result from the binding of different effector molecules to cell surface proteoglycans. It is important to determine the intracellular responses triggered by effector binding and how these responses lead to altered cellular behavior. One way to investigate these matters is to create biological models which are dependent on the expression of specific proteoglycans.

Syndecan-1 is the best characterized cell surface proteoglycan (Saunders et al., J. Cell Biol. 108:1547-1556(1989); Mali et al., J. Biol. Chem. 265:6884-6889 (1990)). It was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan side chains (Rapraeger et al., J. Biol. Chem. 260:11046-11052 (1985)). Recent studies have revealed its expression, not only on epithelial cells but also on differentiating fibroblasts of developing tooth (Thesleff et al., Dev. Biol. 129:565-572 (1988); Vainio et al., J. Cell Biol. 108:1945-1964 (1989)), on endothelial cells of sprouting capillaries (Elenius et al., J. Cell Biol. 114:585-596 (1991)) and on the surface of lymphocyte subpopulations (Sanderson et al., Cell Regul. 1:27-35 (1989)). This suggests that syndecan-1 function can vary from one cell type to another. Syndecan belongs to a family of proteoglycans with conserved plasma membrane and cytoplasmic domains but with dissimilar ectodomains (Mali et al., J. Biol. Chem. 265:6884-6889 (1990)). The conserved structure of syndecans suggests that it could participate in signal transduction through the plasma membrane.

Syndecan-1 binds several extracellular effector molecules but does so in a selective manner. For example, syndecan binds interstitial collagens and fibronectin but does not bind vitronectin or laminin (Koda et al., J. Biol. Chem. 260:8156-8162 (1985)); Saunders et al., J. Cell Biol. 106:423-430 (1988); Elenius et al., J. Biol. Chem. 265:17837-17843 (1990)). Moreover, syndecan-1 isolated from tooth mesenchyme has revealed selective binding to tenascin not observed for syndecan from NMuMG cells (Salmivirta et al., J. Biol. Chem. 266:7733-7739 (1991)). This suggests that variations in syndecan glycosylation alters the binding properties of syndecan. Polymorphism of syndecan-1 glycosylation has also been observed in simple and stratified epithelia (Sanderson et al., Proc. Natl. Acad Sci. USA 85:9562-9566 (1988)); but whether these changes also reflect altered ligand recognition by syndecan remains unknown. Syndecan-1 also binds growth factors, such as basic fibroblast growth factor (Kiefer et al., Proc. Natl. Acad. Sci. USA 87:6985-6989 (1990); Elenius et al., J. Biol. Chem. 267:6435-6441 (1992)).

Fibroblast growth factors (FGFs) are a family of heparin-binding peptides comprising 9 known members. Basic fibroblast growth factor (FGF-2 or bFGF) is synthesized by, and acts on various cell types and tissues. In vitro, it is a strong mitogen for cells of mesodermal origin, can modulate cell motility and differentiation, is a potent angiogenic factor, and potentiates neovascularization in vivo (Burgess and Magiac, Ann. Rev. Biochem. 58:575-606 (1989); Mason, Cell 78:547-552 (1994)). Keratinocyte growth factor (FGF-7 or KGF) is produced solely by cells of mesodermal origin. FGF-7 is proliferative for various epithelial cells (Basilico and Moscatelli, Adv. Cancer Res. 59:115-65 (1992); Rubin et al., Cell Biol. Int. 19:399-411 (1995)), and is also an important mediator of hair follicle growth and differentiation (Danilenko et al., Am. J. Pathol. 147:145-54 (1995); Guo et al., Genes & Develop. 10:165-75 (1996)). Both FGF-2 and FGF-7 are involved in wound healing, where they act as both autocrinic and paracrinic factors. In wounded skin FGF-2 is found in fibroblasts and endothelial cells. This growth factor stimulates proliferation of most cell types involved in wound healing, e.g., keratinocytes, fibroblasts, and vascular and capillary endothelial cells (Bennett et al., Am. J. Surg. 165:728-737 (1993)). FGF-7, which is synthesized only by fibroblasts and is induced during wound healing (Wemer et al., Science 266:819-22 (1994); Werner et al., Proc. Natl. Acad. Sci. 89:6898-6900 (1992), and acts as a paracrinic factor on keratinocytes, inducing their proliferation and migration (Bennett et al., supra). FGF-7 is important for normal wound reepithelialization (Werner et al. (1994), supra). However, recent data with FGF-7 knockout mice indicate that KGF may not be required for normal wound healing (Guo et al., Genes & Develop. 10:165-175 (1996).

Growth factors are involved in the initiation, control, and termination processes of wound healing in an autocrinic and paracrinic manner. FGF-2 is produced by fibroblasts and is also found in association with extracellular matrix and basement membranes where it can be released by proteolytic activity. FGF-2 enhances the accumulation and proliferation of fibroblasts, keratinocytes, endothelial cells, and macrophages. In animal models it induces neovascularization, cell migration, and granulation tissue formation. It has been shown to accelerate wound healing in several different situations, e.g., incisions, burns, and diabetic wounds (See Bennett and Schulz, Am. J. Surg. 165: 728-737 (1993)).

Several AP-1 regulated genes are expressed during wound healing. Fos is rapidly activated on the wound healing edge (Martin and Nobes, Mech. Dev. 38: 209-215 (1992)). Jun may also be activated during wounding or wounding induced tumorigenesis (Marshall et al., Virology 188(1): 373-379 (1992). Cancerous cells are also known to be able to activate the AP-1 complex, and c-fos is required for malignant tumor progression (Saez et al., Cell 82:721-732 (1995)).

Yayon and coworkers (Yayon et al., Cell 64:841-848 (1991)) and Rapraeger and coworkers (Rapraeger et al., Science 252:1705-1708 (1991)) have shown that heparin-like molecules are required for the binding of FGF-2 to its high affinity receptor, indicating that syndecan-like molecules can also modulate the growth factor response. It has been observed that heparin is required for oligomerization of FGF-1 molecules leading to FGFR dimerization or further oligomerization and further signaling (Spivak-Kroizman et al., Cell 79:1015-24 (1994); Ornitz et al., Mol. Cell Biol. 12:240-47 (1992)). Several mechanisms, for both negative and positive regulation for FGF action by proteoglycans have been postulated (Schlessinger et al., Cell 83:357-360 (1995)). Syndecan-1 (Saunders et al., J. Cell Biol. 107:1199-1205 (1989)), can simultaneously bind FGF-2 and extracellular matrix molecules and this complex is able to promote DNA synthesis in 3T3 cells (Salmivirta et al., J. Biol. Chem. 267:17606-17610 (1992)).

However, it has also been reported that different heparin sequences can either activate or inhibit FGF-2 function (Guimond et al., J. Biol. Chem. 268:23906-23914 (1993)) and that the composition and length of syndecan side chains vary in a cell and tissue dependent manner (Sanderson and Bemfield, Proc. Natl. Acad. Sci. USA 85:9562-9566 (1989)); Rapraeger, J. Cell Biol. 109:2509-2518 (1989)); Salmivirta et al., J. Biol. Chem. 267:17606-17610 (1991)). Negative regulation of FGF action by syndecan-1 has also been reported (Mali et al., J. Biol. Chem. 268:24251-24258 (1993)); Aviezer et al., J. Biol. Chem. 269:114-121 (1994)), which may be due to the glycosaminoglycan side chain modification or different stochiometric ratios of FGFR and its co-receptor.

FGF-2 induces the transcription of number of genes encoding transcription factors, components of cytoskeleton, and ribosomal components (Burgess and Magiac, supra). For various growth factors and cytokines the inducible downtream transcriptional mechanisms are well characterized.

Several growth factors can elicit immediate early responses after receptor activation. For example, epidermal growth factor (EGF) and platelet derived growth factor (PDGF) induce, via the ras/ERK pathway, the ternary complex factor. The ternary complex factor acts together with the serum response factor to activate the serum response element (SRE) (Hill and Treisman, EMBO J. 14:5037-5047 (1995)). The cAMP response element (CRE) bound by CRE binding protein (CREB) homodimer, or as heterodimers in association with members of the ATF family, are also under the influence of growth factors, e.g., EGF, PDGF, and FGF-2. EGF and PDGF, with various cytokines, are also able to induce activation of signal transducers and activators of transcription (STAT), which are activated by Janus Kinases (JAKs) and act on the sis-inducible element (SIE) or the interferon stimulated response element (IRSE) (Hill and Treisman, Cell 80:199-211 (1995); Karin, Cur. Op. Cell Biol. 6:415-424 (1994)). Transforming growth factor alpha (TGFα) can activate nFkB.

FGF-2 and -7 both signal by binding to different cell surface tyrosine kinase receptors. However, no clear response element has been previously described for FGF and the signal receiving transcription factors remain to be discovered. FGF-2 can also be localized to the nucleus, where it is supposed to activate transcription (Nakiniski et al., Proc. Natl. Acad. Sci USA 89:5216-5220 (1992); Bouche et al., Proc. Natl. Acad. Sci. USA 84:6770-6774 (1987)) as does FGF-1 (Wiedloche et al., Cell 76:1039-1051 (1994)). Accumulating evidence indicates that FGFs activate the MEF-MAP kinase pathway downstream from FGFR activation, via ras and raf (Umbhauser et al., Nature 376:58-62 (1995)). However, other signal transduction pathways also might contribute to FGF signaling, including a phospholipid-C driven pathway (Mason, Cell 78:547-552 (1994)).

Synergistic action of FGF-2 and cAMP by c-Jun and ATF-3 transcription factors at CRE has also been reported (Tan et al., Mol. Cell Biol. 14:7546-7556 (1994)). However, FGF was a poor activator of transcription without cAMP, indicating that besides FGF this type of FGF induced gene activation requires other extracellular signaling molecules. The SER of a-actin promoter, but not the fos promoter, has been proposed to be another target for FGF-2 induced gene activation in a cell culture model of cardiac myocytes (Parker et al., J. Biol. Chem. 267: 3343-3350 (1992)). For FGF induced negative regulation, one established gene element is the E-box motif, which binds myogenic basic helix-loop-helix (bHLH) transcription factors known to be inhibited by FGF-2. Proteins of the MyoD family: MyoD, myogenin, Myf5, and MRF, bind E-box elements in regulatory regions of various myogenic genes and are responsible for the terminal differentiation of myoblasts to myocytes (Edmondson and Olson, J. Biol. Chem. 268:755-758 (1993)).

The FGF-2 signaling effect seems to be dependent on its molecular weight. Different forms of FGF-2 are created in cells by alternative splicing. The low molecular weight form can induce cell migration, but does not have the mitogenic or proliferative potential of the high molecular forms (Bikfalvi et al., J. Cell Biol. 129:233-243 (1995)). The different molecular forms thus seem to use different signaling pathways.

The fact that cell surface proteoglycans can bind both growth factors and matrix components suggests that proteoglycans play a role in regulating, both temporally (timing of expression) and spatially (precise localization), growth promotion by immobilizing effector molecules to the vicinity of cell-matrix interactions. This is supported by the pattern of syndecan-1 expression during development which follows morphogenetic, rather than histological, patterns (Thesleff et al., Dev. Biol. 129:565-572 (1988); Vainio et al., J. Cell Biol. 108:1945-1954(1989); and Vainio et al., Dev. Biol. 134:382-391 (1989)), and by the observation that syndecan expression is localized to sites of active proliferation (Elenius et al., J. Cell Biol. 114:585-596 (1991) and Vainio et al., Dev. Biol. 147:322-333 (1991)).

In simple epithelium, syndecan-1 is polarized to baso-lateral surfaces where it co-localizes with actin rich cytofilaments (Rapraeger et al., J. Cell Biol. 103:3683-2696 (1986)). Upon rounding, syndecan-1 is shed from the cell surface by proteolytic cleavage of the core protein at the cell surface, a process which separates the matrix binding ectodomain from the membrane domain (Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). In this way, syndecan-1 has been proposed to be involved in the maintenance of epithelial morphology. When mouse mammary tumor cells (S115) are induced to change their morphology from an epithelial to a more fibroblastic or fusiform phenotype, syndecan-1 expression is lost (Leppä et al., Cell Regul. 2:1-11 (1991)). This loss has been found to occur in other cell types undergoing transformation (Inki et al., Am. J. Pathol. 139:1333-1340 (1991); Inki et al., Lab. Invest. 66:314-323 (1992)) suggesting that the loss of syndecan-1 expression is a common characteristic of malignant transformation.

SUMMARY OF THE INVENTION

The present invention is directed to a method for altering the differentiated state of a host cell by altering its expression of syndecan-1. The invention is also directed to a method for inducing and regulating syndecan-1 expression, especially in cells which exhibit a malignant phenotype, regardless of the origin of transformation.

In another aspect, the invention is directed to a treatment for suppressing tumor growth in a patient in need of such treatment, by the administration of a composition comprised of efficacious amounts of one or more agents that stimulate syndecan-1 synthesis in the tumor cells of such patient.

The invention is also directed to the DNA encoding enhancer and suppressor elements of the syndecan-1 gene and to the use of these elements for regulating heterologous gene expression. The vectors and host cells which incorporate DNA sequences containing the syndecan enhancer or suppressor are also encompassed by the invention. Methods of expressing a standard or heterologous gene comprising activating the syndecan enhancer element are also provided.

In addition, the invention is directed to a method for enhancing syndecan-1 expression in a host cell by activating the enhancer element of the syndecan-1 gene.

The invention is also directed to a method for enhancing syndecan-1 expression in malignant cells, by preventing suppression of syndecan gene transcription.

The invention is also directed to a biochemical method for the inactivation of suppressors of syndecan-1 gene expression in malignant cells.

In another aspect, the invention is directed to a method for stimulating cellular differentiation by enhancing syndecan-l expression in both malignant and normal cells.

The invention is also directed to a method for stimulating cellular proliferation and differentiation, thus promoting tissue regeneration, especially in processes such as wound healing, by enhancing syndecan-1 expression.

This invention also relates to an enhancer element of the syndecan-1 gene having about 280 nucleotides. In addition, this invention relates to nuclear proteins that bind to the syndecan enhancer element causing enhancer activation, to the binding sites for these nuclear proteins on the syndecan enhancer, and to a complex of these nuclear proteins bound to the syndecan enhancer element. In particular embodiments of this invention, this nuclear protein is FIN-1, AP-1, or USF. In more specific embodiments, one or more of these proteins are in an oligomeric form, such as a dimer.

The invention further relates to a method of targeting expression of a gene product to cutaneous wounds using a construct comprising the syndecan enhancer gene operably linked to the gene of interest. Non-human transgenic animals comprising the syndecan enhancer operably linked to the gene of interest inserted into this germ line are also provided.

In another aspect, this invention relates to a method of activating or enhancing syndecan-1 expression in malignant cells by activating the syndecan enhancer element. In specific embodiments of this invention, the enhancer element is activated by FIN-1.

Further features, objects and advantages of the present invention will become more fully apparent to one of ordinary skill in the art from a detailed consideration of the following description of the subject invention when taken together with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1 is a diagram of the assembly of mouse syndecan-1 gene and its promoter region.

FIGS. 2a- 2 o. FIGS. 2a- 2 o contain the complete sequence of the mouse syndecan-1 gene (SEQ ID No: 1: (DNA) and SEQ ID No: 2: (protein)). Regulatory sites for the expression of syndecan may also exist on the first intron following the first exon (see FIG. 1).

FIG. 3. FIG. 3 is a diagram of the assembly of mouse syndecan promoter region and the localization of the enhancer and suppressor elements together with restriction sites for three different enzymes.

FIG. 4. FIG. 4 is the complete sequence of the mouse syndecan enhancer element [SEQ ID No. 3: (DNA)] located 8-10 kbs upstream from the transcription initiation site as indicated in FIG. 3.

FIGS. 5a-5 d. FIGS. 5a-5 d are a photographic presentation of reduced growth ability of syndecan-transfected cells in soft agar. Panel 5 a (wild-type S115 cells) and 5 b (control transfected cells) are pictures of the colonies that are formed in soft agar in the presence of testosterone, a feature typical for hormone-transformed cells. This growth ability was not observed with two independent syndecan-transfected cell clones (panels 5 c and 5 d), demonstrating how syndecan-1 re-expression can overcome the effect of hormone-induced transformation.

FIG. 6. FIG. 6 is a graphical presentation of how syndecan-transfected cells lose their ability to form tumors in nude mice. Wild-type or control transfected cells produce tumors in testosterone-administered nude mice while syndecan transfected cells revealed a very low tendency to produce tumors.

FIG. 7. FIG. 7 is a graphical representation of enhanced syndecan-1 expression in 3T3 cells by simultaneously administered basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-β). This is an example of how syndecan-1 expression can be enhanced as a result of growth factor action in normal cells during the differentiation process.

FIG. 8. FIG. 8 is a graphical representation of enhanced syndecan-1 expression by MCF-7 cells exposed to the anti-estrogen toremifene. When exposed to estrogen, syndecan-1 expression in MCF-7 cells was reduced and the cells transformed. Subsequent treatment with the anti-estrogen (toremifene) restored syndecan-1 expression to levels close to that found in cells not exposed to estrogen and aided the cells in maintaining their normal growth behavior.

FIGS. 9a and 9 b. FIG. 9 is a graphical presentation of how the suppressor element (see FIG. 3) is active in S115 cells treated with testosterone. Indicated stretches of promoter sequences were transfected in hormone-treated S115 cells and analyzed for their transcription activity as described in Example VI. A dramatic drop was observed with suppressor construct as indicated in FIG. 3, and it was more obvious in transformed S115 cells (FIG. 9a) than in 3T3 cells (FIG. 9b). The vertical axes shown in the figure represent percent expression wherein expression in the absence of suppressor is taken as 100%.

FIG. 10. FIG. 10 is a graphical presentation of how the enhancer element is active in growth hormone-treated 3T3 cells. Various stretches of promoter were transfected in 3T3 cells and analyzed for their transcription activities. Fragment pXb6, which is the same as illustrated in FIG. 3 as an enhancer, revealed more than a ten fold stimulation of expression in 3T3 cells exposed to growth factors bFGF and TGFβ if compared to non-treated cells. The vertical axis of the figure represents percent expression wherein the expression observed in untreated cells is taken as 100%.

FIG. 11a. FIG. 11a depicts the activation of the syndecan-1 gene by FGF-2 in NIH3T3 cells. 3T3NIH cells were exposed to 10 ng/ml of FGF-2 for 0, 4, 6, 8, 12, 24 and 48 hrs (F0-F48). RNA was isolated and the level of mouse syndecan MRNA (Syn-1) was quantified. GAPDH mRNA was included in each sample as a loading control. Control samples, with no FGF-2 treatment, were assayed for Syn-1 mRNA at 6,12 and 24 hrs (C6-R4). Two individual exposures for each time point are depicted.

FIG. 11b. FIG. 11b shows that FGF-2 induced transcription of the syndecan-1 gene is based on the activation of a far upstream enhancer. In FIG. 11b transfection constructs comprising different fragments of the 11.5 kb upstream regulatory region of the syndecan-1 gene operably linked to a 1.1 kb sequence containing the proximal promoter of the syndecan-1 gene the chloramphenicol acetyltransferase (CAT) structural gene were assayed for CAT activity by transient transfection in 3T3NIH cells with and without FGF-2 treatment. Only the construct containing the most distal 2.2 kb fragment (pXb6) showed increased CAT activity following FGF-2 treatment.

FIG. 11c. In FIG. 11c this FGF-2 inducible region from pXb6 was further subdivided and the fragments were assayed similarly for enhancement of CAT expression. A construct containing a 280 bp element (pFiRE) retained full enhancement of CAT activity following FGF-2 treatment in both the forward (pFiRE) and the reverse orientation (pFiRErev).

FIG. 11d. In FIG. 11d, 3T3NIH cells were exposed to 10 ng/ml of FGF-2, for 4 and 24 hours followed by isolation of nuclei for a run-off experiment. c-jun and nur transcription was used as a positive control, and β-actin as a loading control.

FIGS. 12a-12 c. FIGS. 12a-12 c shows that enhancer activation is specific for FGF-2 in NIH3T3 cells. pFiRE was used to transfect 3T3NIH cells as in FIGS. 11a-11 d. The cells were starved in DMEM regular medium supplemented with CMS-serum (growth factor depleted) for 1 day. The transformed cells were then exposed to various growth factors in the same medium or to 5% fetal calf serum (FCS) supplemented medium. FGFs showed a clear enhancer activation not seen with the other growth factors that were tested (FIG. 12a). However, all the growth factors that were treated increased DNA synthesis, as tested by a ¹²⁵IdU incorporation assay (FIG. 12b). The removal of the syndecan promoter had no effect on the pattern of growth factor induced FiRE activation (FIG. 12c).

FIG. 13a. FIG. 13a shows the results of DNAse I footprinting experiments of the enhancer element, indicating that the enhancer includes five DNA binding motifs. DNAse I footprinting was performed with end-labeled pFiRE vector alone (naked) or together with FGF-2 induced (FGF-2) and non-induced (control) nuclear extracts. The products of the A+G sequence reaction of pFiRE are also depicted. Five footprinted motifs, showing binding of proteins in the nuclear extract to sequences within pFiRE, are marked with numbered boxes.

FIG. 13b. FIG. 13b depicts the sequence of the FGF-2 inducible enhancer. The sequence of the pFiRE fragment is shown. The sequence of the footprinted motifs identified in FIG. 13a are underlined and together restore 100% of the enhancer activity. Motif 3 contains the binding site for FGF-inducible nuclear factor FIN-1. This binding site does not contain sequences previously identified as consensus binding sites for transcription factors. Motifs 5 and 4 are AP-1-like binding sites, motif 2 is an E-box, and motif 1 is an unknown transcription factor recognition site.

FIGS. 14a-14 f. FIGS. 14a-14 f shows that FGF-2 inducible and non-inducible nuclear factors bind to the enhancer. In FIG. 14a, the results of a gel retardation analysis for each enhancer motif and for an SP1 consensus oligonucleotide (control) are shown. This analysis was performed to obtain protein complexes that bind at each of the protein binding site shown in FIG. 14. Nuclear extracts derived from FGF-2 treated (f or FGF) or untreated (c or control) 3T3 cells were incubated with DNA containing motifs 1-5 as indicated. In FIGS. 14b-14 f, motifs 1 (FIG. 14b), 2 (FIG. 14c), 3 (FIG. 14d), 4 (FIG. 14e), or 5 (FIG. 14f) were further competed with a molar excess of specific (s) or non-specific (ns) oligonucleotides. The non-specific oligonucleotide for motifs 3, 4 and 5 was the motif 1 sequence and for motifs 1 and 2, the non-specific oligonucleotide was the motif 3 sequence. Specific binding is indicated by an arrow.

FIGS. 15a-15 f. FIGS. 15a-15 f show that FiRE involves FGF-inducible AP-1s and constitutive USFs. In FIG. 15a, specific binding (indicated by a line) on motif 5 was competed by molar excess of AP-1 consensus oligonucleotide, but not by E-box consensus oligonucleotide. Antibodies against c-fos and cjun removed the specific bond and raised a supershift (arrow). Antibodies against USF, used as a negative control, had no effect.

FIG. 15b. In FIG. 15b, specific binding (indicated by a line) on motif 4 was competed by a molar excess of AP-1 consensus oligonucleotide, but not by E-box consensus oligonucleotide. Antibodies against c-fos and c-jun removed the specific bond and raised a supershift (arrow). Antibodies against USF, used as a negative control, had no effect.

FIG. 15c. In FIG. 15c specific binding (indicated by a line) on motif 3 was competed by a molar excess of AP-2 consensus oligonucleotide. AP-2 antibody raised a supershift in a reaction with labeled AP-2 oligonucleotide and 3T3 nuclear cell extracts but did not produce a supershift with labelled motif 3 and nuclear extracts deemed from FGF-2 treated 3T3 NIH cells. A gel retardation gel as shown in FIG. 15a was run and exposed to UV light. The specific bands, representing the bound protein-DNA complex, were cut out, eluted overnight, and loaded onto an SDS-PAGE gel to analyze their molecular weight. Two reproducible bands for the motif 3 binding protein are shown. The molecular weight of the nuclear factors were approximated by subtracting the calculated molecular weight of each oligonucleotide from the complex molecular weight.

FIG. 15d. In FIG. 15d, specific binding (indicated by a line) on motif 2 was competed by a molar excess of E-box consensus oligonucleotide, but not by AP-1 consensus oligonucleotide. Antibody against USF eliminated the specific band, but the c-jun antibody had no effect.

FIG. 15e. In FIG. 15e the molecular weight of the protein bound to motif 1 is determined following UV crosslinking.

FIG. 15f. FIG. 15f is a schematic depicting the binding of various nuclear factors to the syndecan enhancer element.

FIG. 16. FIG. 16 shows that enhancer activity is reduced in 3T3 cells expressing high levels of syndecan-1. Syndecan-1 transfected (3T3 1.5) and wild-type (3T3 WT) 3T3 cells were transiently transfected with the pFiRE construct and analyzed for reporter gene expression (CAT activity). The pFiRE activation observed in 3T3 cells not expressing high levels of syndecan-1 was significantly reduced in syndecan-1 expressing 3T3 cells. Solid bars indicate CAT activity in non-treated cells, while hatched bars indicate CAT activity in FGF-treated cells.

FIG. 17. FIG. 17 is a plasmid map of pTRG Syn Prom, showing various restriction sites within the MCS-SP-LacZ region.

FIG. 18. FIG. 18 is a plasmid map of pTRG ENH, showing various restriction sites within the MCS-SP-LacZ region.

FIGS. 19a-f. FIGS. 19a-f depicts micrographs showing staining resulting from reporter gene activation in 2 day old transgenic mice tail wounds. FIG. 19a is a schematic presentation of the transgenic constructs used. The SynPro construct contains 2.3 kb of the syndecan regulatory region from the XhoI restriction site (+140 bp from the major transcription initiation site) to SphI (−2.2 kb) operably linked to the β-galactosidase reporter gene. The FIRE construct also contains the 280 bp for upstream inducible element (PstI-StyI fragment) in front of SynPro. FIG. 19b depicts a 2 day old wound on SynPro transgenic animal's tail skin showing no X-gal staining. FIG. 19c depicts similar wounds made to FiRE transgenic animal's tails. Heavy staining is seen both in proliferating and migrating keratinocytes and in the keratinocytes of hair follicles close to the wound site (arrow). FIG. 19d shows stratifying epithelial cells closer to the wound edge, where FiRE is still active. FIG. 19e shows closed and recently healed wounds, where the staining is fading. FIG. 19f shows resting skin where FiRE is not activated.

FIGS. 20a-20 c. FIGS. 20a-20 c show the results of cell culture experiments demonstrating that FiRE is specific for FGF-7 in keratinocytes.

FIG. 21. FIG. 21 depicts the amount of enhancer activation, as measured by the relative expression of the CAT reporter gene, for deletion mutants of each motif. For the motif 1 deletion mutant (Del M1), the 3′ end of FIRE was deleted. For the motif 2 deletion mutant (Del M2), the E-Box was replaced by a KpnI recognition site. For motif 3-4 deletion mutants to (Del M3 and Del M4), a central 10 bp sequence was replaced with a 10 bp sequence including an SpeI recognition site. For the motif 5 deletion mutant (Del M5) a PCR product from motif 4 to motif 1 was generated. The mutant enhancers were ligated into a CAT reporter plasmid with a 1.1 kb sequence proximal promoter and transfected. Cells were treated with FGF-2 and CAT activity was assayed.

FIGS. 22a-22 d. FIGS. 22a-22 d depict the results of experiments to determine whether protein synthesis is needed for the induction of FGF-inductible transcription factors. Cells were treated with FGF-2 with our without 1 mg/ml (final concentration) of translation inhibitor cycloheximide. Each motif (2-5) is shown with separately run control (Cont.), FGF-2 (FGF), and simultaneous FGF-2 and cycloheximide treatment (FGF+cycloh.). The cycloheximide treatment abolishes the specific binding on motifs 3, 4, and 5, but not on motif 2.

FIG. 23. FIG. 23 depicts the amount of enhancer activation, as measured by the relative expression of the CAT reporter gene, for pXb6 containing the SV40 promoter and pCATSynProm in the presence and in the absence of the simultaneous administration of FGF-2 and TGF-β.

FIG. 24. FIG. 24 depicts experiments showing that TPA or other activators of AP-1 do not activate the AP-1 driven FiRE. Different agents known to induce fos-jun complexes and activate AP-1 driven promoters, were tested for FiRE activation in CAT assay with the minimal promoter transferred in stable 3T3NIH cells in the presence of protein kinase C activator (TPA, 10 mm), protein phosphatase 1 and 2 inhibitors (DA and calycalin A), a cAMP activator (forskolin), or a tyrosine phosphatase inhibitor (orthovanadate).

DEFINITIONS

In order to provide a clearer and more consistent understanding of the invention the following definitions are provided.

“Enhancement” or “Stimulation” of syndecan expression. By “‘enhancement’ or ‘stimulation’ of syndecan expression” is meant an effect of increasing the synthesis of syndecan, either by the induction or de-suppression (de-repression) of syndecan gene transcription and/or translation.

Cell growth. By “cell growth” is meant cell replication, both controlled and uncontrolled.

Malignant. By “malignant” is meant uncontrolled cell growth.

More Differentiated Phenotype. In stating that a cell has a “more differentiated phenotype,” it is meant that the cell possesses a phenotype usually possessed by a certain cell type more differentiated than the cell, which the cell was deficient in prior to enhancement of syndecan expression. This phenotype may be defined by one or more phenotypic characteristics. For example, an epithelial cell is a more differentiated phenotype of a mesenchymal-like shape; therefore, the ability of the method of the invention to maintain cells in an epithelial cell morphology rather than a mesenchymal-like shape is a more. differentiated phenotype within the meaning of the definition. Continuous syndecan expression is necessary for the maintenance of terminal differentiation of epithelial cells.

Syndecan expression is also linked to the normal differentiation of mesenchymal cells. However, unlike epithelial cells, continued expression of syndecan is not needed for maintenance of terminal differentiation in mesenchymal cells. To induce differentiation of suitable mesenchymal precursors (such as a “condensing mesenchymal” cells) to fully differentiated mesenchymal cells, only a transient expression of syndecan is required. A terminally differentiated mesenchymal cell is a “more differentiated phenotype” than a condensing mesenchymal cell.

Other phenotypes that are characteristic of syndecan-deficient cells but not of their non-deficient counterparts include fusiform shapes with long filopodial extensions. There is an extensive under- and overlapping of these processes causing the cells to appear to have a defect in cell adhesion.

In another example, syndecan-deficient NMuMG cells continue to secrete milk fat globule antigen (and thus appear mammary-like) and continue to express cytokeratins (thus appear epithelial-like). However, their actin-containing cytoskeleton is disorganized and their expression of beta, integrins and E-cadherins at the cell surface is markedly reduced. Upon increased expression of syndecan, these phenotypes are corrected so that there is no reduction in cell surface integrins or E-cadherin and the cell has an epithelial morphology. Therefore, the amount of cell surface integrins or E-cadherin is useful as a marker of syndecan expression and may be used to monitor the amount of a drug needed for efficacious results according to the method of the invention.

Efficacious Amount. An “efficacious amount” of an agent is an amount of such agent that is sufficient to bring about a desired result, especially upon administration to an animal or human.

Administration. The term “administration” is meant to include introduction of agents that induce syndecan expression into an animal or human by any appropriate means known to the medical art, including, but not limited to, injection, oral, enteral and parenteral (e.g., intravenous) administration.

Pharmaceutically Acceptable Salt. The term “pharmaceutically acceptable salt” is intended to include salts of the syndecan-inducing agents of the invention. Such salts can be formed from pharmaceutically acceptable acids or bases, such as, for example, acids such as sulfuric, hydrochloric, nitric, phosphoric, etc., or bases such as alkali or alkaline earth metal hydroxides, ammonium hydroxides, alkyl ammonium hydroxides, etc.

Pharmaceutically Acceptable Vehicle. The term “pharmaceutically acceptable vehicle” is intended to include solvents, carriers, diluents, and the like, which are utilized as additives to preparations of the syndecan-inducing agents of the invention so as to provide a carrier or adjuvant for the administration of such compounds.

Treatment. The term “treatment” or “treating” is intended to include the administration of compositions comprising efficacious amounts of syndecan-inducing agents to a subject for purposes which may include prophylaxis, amelioration, prevention or cure of a medical disorder, suppression of tumor growth, or the promotion of hair growth.

Operably Linked. In the context of an expression vector, “operably linked” refers to a location, juxtaposition, and/or orientation of various elements, e.g., enhancer, promoter, and structural gene, within an expression vector so that the structural gene is expressed in any appropriate host cell. When an enhancer is operably linked to a structural gene, expression of the structural gene is enhanced over the level of expression observed in the absence of the enhancer.

Syndecan Enhancer Element. In the context of this invention, “syndecan enhancer element” is a DNA sequence having significant structural homology to the sequence of FIG. 13b and retaining the same functional properties as the sequence of FIG. 13b, e.g., enhancing the expression of the syndecan-1 gene by FGFs. For example, in certain embodiments of this invention, a syndecan enhancer element may retain 95% sequence homology to the sequence in FIG. 13b, while retaining enhancer activity. In other embodiments of this invention, a syndecan enhancer element may retain 90% sequence homology to the sequence in FIG. 13b, while retaining enhancer activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Briefly, in its broader aspects, the present invention comprehends a method for maintaining a differentiated phenotype in a normal (non-malignant) cell that otherwise would suppress syndecan-1 expression, by maintaining syndecan-1 expression in such cell. The invention also comprehends a method for inducing a more differentiated phenotype in a malignant cell that lacks (or is deficient in) syndecan-1 expression, by stimulating syndecan-1 expression in such a cell. As used herein, a cell said to “lack” syndecan-1 expression, may either be completely deficient in syndecan-1 protein or produce insufficient syndecan-1 to maintain or attain a desired differentiated phenotype.

The methods of the invention will not only prevent the progression (worsening) of a transformation state and the growth of tumors, but will also maintain cells in a state in which they continue to perform differentiated functions. Examples of differentiated functions of non-malignant cells include the secretion of specific proteins and/or other macromolecules and hair formation by epidermal cells of skin. Thus, according to the invention, administration of agents capable of inducing syndecan-1 expression in epidermal skin cells of the scalp will promote hair growth among bald (or balding) people.

The subject method may be accomplished by biochemical, chemical or even molecular biological methods. While the method is applicable to a variety of cancer (both malignant and non-malignant) and normal cells, it is particularly adaptable for treating malignant cells which have become transformed. This includes cells transformed due to hormonal influences of the body or environmental influences, such as chemicals or radiation exposure. The method is especially effective for tumors characterized by loss of syndecan-1 expression, for example, a glioma, myeloma, carcinoma, sarcoma, lymphoma, or adenoma.

Generally, any cell genetically capable of expressing syndecan-1 can be stimulated to express syndecan-1 by the method of the invention. Syndecan-1 is naturally expressed in a wide variety of epithelial cells in mature and embryonic tissues and by various embryonic mesenchymal tissues undergoing inductive interactions with epithelia. In addition, syndecan-1 is naturally expressed by Leydig cells, by developing B-lymphocytes and by a subpopulation of plasma cells.

Enhanced syndecan-1 expression may be achieved by administration of compositions containing a biochemically, and/or chemically and/or molecular-biologically active component to an individual. Compositions may be administered orally, intravenously, subcutaneously or locally, or by any other method which will allow cells, normal or malignant, to be exposed to the syndecan-1 expression enhancing component.

By a “biochemically” or “chemically” active component is meant a component that alters the endogenous syndecan biochemistry or chemistry of the target cell without altering syndecan gene expressionper se. Such alteration may include altering the half-life of syndecan protein or mRNA, so as to increase levels of syndecan-1 protein in the cell. For example, by altering the external domain of the cell's endogenous syndecan, or its cell surface membrane properties in general, may be altered so as to retain higher levels of syndecan on the cell surface; and, altering the syndecan protein active site(s), so as to enhance the efficiency of the syndecan response.

By a “molecular-biologically” active component is meant a component that alters endogenous syndecan-1 gene expression in a manner that allows for an increase in cellular syndecan-1, such as, for example, by stimulating transcription, preventing (or reducing) suppression of transcription, de-repression of transcription, or by generally increasing levels of mRNA and/or translation efficiency.

It is known that cellular transformation involves activation of cellular growth-stimulating genes (e.g., oncogenes) and inactivation of other genes which suppress cell growth. It has recently been shown that loss of syndecan-1 expression is observed upon transformation of cells, and that this suppression is due to syndecan gene inactivation (Leppä et al., Cell Regul. 2:1-11 (1991); Inki et al., Am. J. Pathol. 139:1333-1340 (1991); Inki et al., Lab. Invest. 66:314-323 (1992)). This was demonstrated in several biological models of carcinogenesis including models in which transformation is caused by oncogenes, by chemical carcinogens, by UV-light or by hormone-exposure. Thus, syndecan-1 gene suppression is a general phenomenon associated with cellular transformation. All the manipulations of such cells which induce syndecan-1 expression cause these cells to assume a more differentiated phenotype, and thus, reduce their potential tumorigenic behavior and tendency to metastasize.

In order to determine whether steroids were having a direct and causal effect on the transformation of S115 cells, normal regulatory elements of the syndecan-1 gene were replaced with hormone-inducible elements. As a result of this change, the new cell lines no longer underwent transformation as the result of exposure to steroid.

In a preferred embodiment, the cell in which syndecan-1 expression is stimulated is steroid-responsive. Examples of such steroid-responsive cells include breast cells, endometrium cells and prostate cells, especially in the malignant state. In a highly preferred embodiment, the cell is responsive to estrogen and/or androgen.

Examples of other cell types that will respond to the treatment of the invention include malignant and non-malignant mesenchymal cells.

The regulatory elements of a given gene are commonly located upstream from (i.e., 5 prime to) the transcription initiation site. Syndecan-1, however, has a very peculiar gene structure, in which the first and second exons are separated by a very large intron (FIG. 1). This could mean that, in addition to base sequences upstream from the transcription site, syndecan expression may also be susceptible to regulation by base sequences located in the first intron.

Nucleotide sequence elements responsible for regulating syndecan gene expression were identified by ligating DNA elements lying upstream of the syndecan gene transcription initiation site to vectors containing the chloramphenicol acetyltransferase (CAT) gene and determining the CAT enzymatic activity expressed by cells transformed with such vectors (see Example VI for experimental details).

It was found that the syndecan-1 gene has a strong enhancer element located approximately 11.5 kb upstream from the transcription initiation site. Results such as those presented in FIG. 10 indicate that the DNA element shown in FIG. 4 (SEQ ID No. 3) is capable of enhancing expression. Further experiments localized the enhancer to the 350 nucleotide sequence shown as SEQ ID No. 4. DNA encoding the enhancer may be linked to recombinant constructs containing a promoter and a structural gene and may serve to enhance recombinant expression.

The enhancer element has now been further localized to a 283 nucleotide sequence, depicted in FIG. 13b (SEQ ID NO. 5). This sequence may also be linked in either orientation to recombinant constructs containing a promoter and a structural gene, including heterologous promoters or structural genes, to enhance recombinant expression.

Cells destined to differentiate during organ formation or tissue regeneration also exhibit enhanced syndecan-1 expression (Vainio et al., Dev. Biol. 147:322-333 (1991); Elenius et al., J. Cell Biol. 114:585-595 (1991)). Growth factors are candidates for regulating syndecan-1 expression since they are known to be involved in the regulation of early development and cellular differentiation (Heath et al., Curr. Opin. Cell Biol. 3:935-938 (1991)). The suggestion that growth factors are involved is also supported indirectly by the fact that the expression of two embryonically important growth factors (TGF-β and FGF) has been shown to coincide with syndecan-1 expression in developing tooth (Vaahtokari et al., Development 113:985-994 (1991); Wilkinson et al., Development 105:131-136 (1989)).

Based upon these findings, the possible effect of growth factors on the expression of syndecan-1 has been tested. It was found that both FGF-2 and TGF-β enhance syndecan-1 expression by 3T3 cells, if these agents are administered together (FIG. 7). FGF-2, when administered alone also shows enhancement of syndecan-1 expression. This stimulation produced syndecan-1 levels close to the levels observed in syndecan-expressing epithelial cells (Elenius. et al., J. Biol. Chem. 267:6435-6441 (1992)) prior to their becoming malignant (Leppä et al., Proc. Natl. Acad Sci. USA 89:932-936 (1992)). The findings suggest that growth factors, and their derived fragments and domains may prove to be valuable tools for the regulation of syndecan expression.

This 280 bp FGF-inducible response element is located 11.6 kb from the translation start site of the syndecan-1 gene and shows at least about a ten-fold activation in FGF-2 treated 3T3 cells. In mesenchymal cells this activation seems to be restricted to FGF-2. No response to serum or other growth factors tested was observed. Interestingly, FiRE also shows a distinctive specificity among the FGF family members, because FGF-1 did not activate or suppress this element. This suggests a separate signaling pathway and function for FGF-1 and -2 in mesenchymal cells. FGF-7 is a poor activator of mesenchymal cells and did not activate the enhancer. However, in cultured keratinocytes FGF-7 was able to activate FiRE. However, serum, PDGF, FGF-1 and surprisingly also FGF-2, failed to activate FiRE in cultured keratinocytes. In another epithelial cell line (NMuMG) FiRE was not inducible by any of the growth factors tested, which was expected, since NMuMG are FGFR negative cells. This indicates that the activation FiRE is cell specific, and in addition, is selective for different members among the FGF family in a cell dependent manner.

FiRE binds both FGF inducible and non-inducible nuclear factors bound in an organized array (see FIG. 15f). The FiRE nuclear factors include two AP-1 factors, which are homo- or heterodimers composed of the products from the fos and jun families. Members of thefos and jun families include c-Jun, JunB, JunD, c-fos, Fos-B, Fra-1 and Fra-2. Components of AP-1, fos and jun, as well as other members of the fos and jun families, are rapidly activated by a vast amount of extracellular stimuli. Their activation is brought about by either direct gene activation or posttranslational modifications (Karin, Curr. Op. Cell Biol. 6:415-424 (1994)). In several studies, FGFs are shown to activate c-fos. FGF-2 is also known to induce the expression of c-fos and c-jun in 3T3 cells (Janet et al., Exp. Cell Res. 198:305-344 (1992)). Our results show that FGF treatment results in transcriptionally active fos-jun heterodimers. FiRE is bound by at least two AP-1 complexes, which both seem to be required for FGF induction, since deletion of one AP-1 site dramatically decreases the FGF response.

FiRE also binds USF, which is a ubiquitously expressed homodimeric transcription factor (Gregor et al., Genes Develop. 4:1730-1740 (1990)). The binding site for USF on the FiRE is the E-box. USF is constitutively expressed in 3T3 cells (Miltenberger et al., Mol. Cell Biol. 15:2527-2535 (1995)). It is not known to be under influence of any growth factor. In FiRE, USF is not activated by FGF but it is involved in the complex with inducible components. Replacement of the E-Box does not, however, strongly reduce the FGF response on FiRE. This might be due to replacement of USF with another constitutively active transcription factor, which would be able to interact with inducible components. Alternatively, protein-protein interactions might hold USF in the FiRE complex although its optional binding target in DNA has been destroyed. However, the integration of the FiRE-reporter gene construct into the chromosome could still result in requirement for USF since these experiments were done with extrachromosomal plasmid DNA in a transient transfection.

Besides AP-1 and USF the FiRE element also binds another non-inducible component as well as a novel FGF-inducible AP-2-like transcription factor. AP-2 is a cell-specific 50 kDa transcription factor expressed, among other tissues, in keratinocytes in high amounts (Leask et al., Proc. Natl. Acad. Sci USA 88:7948-7952 (1991)). Multiple forms of AP-2 are generated by alternative splicing (Meier et al., Dev. Biol. 169:1-14 (1995)) and also one AP-2 related transcription factor, AP-2b has been cloned (Moser et al., Development 121:2778-2788 (1995)). FIN-1 is a novel FGF-inducible nuclear factor that binds FiRE and can be competed with AP-2 consensus oligonucleotide. This factor is not recognized by AP-2 antibody, but could be a member of a larger AP-2 family. It is known, that AP-2 can form heterodimers without DNA binding and, that AP-2 dimerization is required for binding (Williams and Tijan, Science 251:1067-1071 (1991)). Thus, FIN-1 likely binds to FiRE as a dimer (see FIG. 15).

The complex structure of FiRE with its far upstream location could explain the difficulties of discovering FGF induced gene activation. It also indicates that FGF-induced signals are not restricted to CRE and SRE, their formerly proposed endpoints.

FiRE can be considered a secondary reponse element requiring de novo protein synthesis after initial FGFR activation. This response element can further diversify FGF influence on different cell types. FiRE is the first example of this type of a response element.

The binding sites for each of these nuclear proteins on the enhancer element have been mapped, see FIGS. 14a-14 f, and their sequence has been identified. See FIGS. 15a-14 f. AP-1 binds to motifs 4 and 5; FIN-1 binds motif 3; USF binds motif 2; and p46 binds motif 1. These binding sites constitute 100% of the enhancer activity. These binding sites on the enhancer element for FIN-1 do not contain any previously described consensus sequences for transcription factors.

Thus the invention is also directed to a recombinant DNA molecule that comprises a syndecan enhancer element that comprises each of the following sequences:

(a) TGGCACACCCACCGTCACGAGAGCT (SEQ ID No. 11);

(b) TGGCACACCTGGGAG (SEQ ID No. 12);

(c) TGGTTCAGGGTGACT (SEQ ID No. 13);

(d) AGGAGTGAGCCATGCCACC (SEQ ID No. 9); and

(e) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG (SEQ ID No. 14).

Alternatively, (a) can be dGCTGGCACACCCACCGTCACGAGAGCTTCC (SEQ ID No. 6), (b) can be TTGGCACACCTGGGAGGATG (SEQ ID No. 7), (c) can be AGTGGTTCAGGGTGACTCT (SEQ ID No. 8), and (e) can be CTGGGTCATTGATGACTG (SEQ ID No. 10).

The invention is also directed to a recombinant DNA molecule that comprises a syndecan enhancer element that comprises at least one sequence selected from the group consisting of:

(a) TGGCACACCCACCGTCACGAGAGCT (SEQ ID No. 11);

(b) TGGCACACCTGGGAG (SEQ ID No. 12);

(c) TGGTTCAGGGTGACT (SEQ ID No. 13);

(d) AGGAGTGAGCCATGCCACC (SEQ ID No. 9); and

(e) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG (SEQ ID No. 14).

Alternatively, (a) can be dGCTGGCACACCCACCGTCACGAGAGCTTCC (SEQ ID No. 6), (b) can be TTGGCACACCTGGGAGGATG (SEQ ID No. 7), (c) can be AGTGGTTCAGGGTGACTCT (SEQ ID No. 8), and (e) can be CTGGGTCATTGATGACTG (SEQ ID No. 10).

The invention is also directed to a recombinant DNA molecule that comprises a syndecan enhancer element that comprises at least one sequence selected from the group consisting of:

(a) TGGCACACCCACCGTCACGAGAGCT (SEQ ID No. 11);

(b) TGGTTCAGGGTGACT (SEQ ID No. 13);

(c) AGGAGTGAGCCATGCCACC (SEQ ID No. 9); and

(d) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG (SEQ ID No. 14).

Alternatively, (a) can be dGCTGGCACACCCACCGTCACGAGAGCTTCC (SEQ ID No. 6), (b) can be TTGGCACACCTGGGAGGATG (SEQ ID No. 7), (c) can be AGTGGTTCAGGGTGACTCT (SEQ ID No. 8), and (e) can be CTGGGTCATTGATGACTG (SEQ ID No. 10).

The invention is also directed to a DNA molecule selected from the group consisting of one or more of the following sequences:

(a) TGGCACACCCACCGTCACGAGAGCT (SEQ ID No. 11);

(b) TGGTTCAGGGTGACT (SEQ ID No. 13);

(c) AGGAGTGAGCCATGCCACC (SEQ ID No. 9); and

(d) CTGGGTCATTGATGACTGTTGTGTGGGATACCTGCCGGG (SEQ ID No. 14).

Alternatively, (a) can be dGCTGGCACACCCACCGTCACGAGAGCTTCC (SEQ ID No. 6), (b) can be AGTGGTTCAGGGTGACTCT (SEQ ID No. 8), and (d) can be CTGGGTCATTGATGACTG (SEQ ID No. 10).

The invention is also directed to a recombinant DNA molecule having the following sequence TGGCACACCTGGGAG (SEQ ID No. 12).

The FIN-1 protein has been isolated and has a molecular weight of 50 kDa as determined by SDS-PAGE.

In addition to activating the syndecan promoter, this enhancer element has been shown to activate the SV40 promoter. The effect of the enhancer element on the SV-40 promoter can be seen in FIG. 21, where activation by the simultaneous administration of FGF-2 and TGF-β is observed. Therefore, FiRE is not syndecan-1 restricted and can be used with any suitable eukaryotic promoter, e.g., SV 40 promoter.

Several other growth factors were tested for the ability to activate the syndecan enhancer in pFiRE transfected 3T3NIH cells. FGF-2 showed clear enhancer activation. However, no significant enhancer activation was shown under these conditions with FGF-2, FGF-7, EGF, PDGF, or TGF-β. Other growth factors could be tested in a similar fashion. 5% fetal calf serum showed a slight stimulation of the enhancer. See FIGS. 12a-12 c. However, in cells expressing high levels of syndecan-1, enhancement activation is only slightly evident, as depicted in FIG. 16.

FCS, FGF-1, FGF-2, FGF-7, EGF, PDGF, and TGF-β were also tested for an increase in DNA synthesis by a 125-IdU incorporation assay. All showed a significant increase in DNA synthesis as represented in the bottom panel of FIGS. 12a-12 c.

This invention also relates to transgenic non-human animals containing the syndecan enhancer operably linked to a promoter and to a structural gene inserted into this germ line. For example, transgenic mice were made containing the syndecan enhancer operably linked to the syndecan-1 proximal promoter and to β-galactosidase functioning as a reporter gene. The first plasmid, pTRGSynProm, contains a 2.25 kb XhoI-StuI restriction fragment comprising the syndecan-1 basic promoter region ligated to the β-galactosidase gene. The second plasmid, pTRGEnh, also contains the 2.25 kb XhoI-StuI restriction fragment ligated to the β-galactosidase gene. However, pTRGEnh also contains an additional 280 bp PstI-StuI restriction fragment containing the syndecan-1 enhancer inserted next to the basic promoter. Plasmid pTRGSynProm is depicted in FIG. 17, while pTRGEnh is depicted in FIG. 18.

These plasmids were transferred into pronuclei of fertilized mouse (FVB/N) oocytes by microinjection and the oocytes were transplanted into pseudopregnant mice (Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Lab. (1986)). Other transgenic animals can be made by appropriately modifying these procedures. For example, rat and bovine transgenic animals can be made. In the transgenic animals containing the syndecan enhancer, reporter gene activity was localized to wounds. Therefore, the enhancer element is associated with the targeting and/or differential expression of the target gene to wounds. By linking the syndecan enhancer to a gene of interest in transgenic animals expression of that gene can be targeted to wounds. Suitable promoters that can be incorporated into these constructs include the syndecan-1 promoter, the thymidine kinase promoter, the keratin-6 promoter, and the keratin-16 promoter. In particular embodiments of this invention, the gene of interest codes for a therapeutic, and the expression of this therapeutic at wound sites can be accomplished using an expression system containing the syndecan enhancer. Examples of suitable genes of interest include EGF and KGF.

Other experiments revealed the existence of a sequence element capable of suppressing gene expression (see FIGS. 9a-9 b). The suppressor was localized to nucleotides between −250 and −600 as shown in FIG. 2(b) (SEQ ID No. 1). DNA encoding the suppressor may be linked to recombinant constructs containing a promoter and a structural gene and may serve to suppress recombinant expression. Alternatively, methods may be devised for blocking the suppressive effect of this element in order to promote syndecan expression.

Manipulation of the upstream region of the syndecan gene can block its inactivation during malignant transformation. For example, replacement of the region in front of first exon of the syndecan gene with the glucocorticoid-inducible elements of mouse mammary tumor virus (MMTV) not only blocks syndecan suppression during malignant transformation, but also inhibits the ability or potential of cells to transform and become tumorigenic (FIGS. 5a-5 d and 6). These findings suggest a very important role for syndecan in the maintenance of normal epithelial morphology (Leppä et al., Proc. Natl. Acad Sci. USA 89:932-936 (1992)).

Preferably, for treatment of humans and animals, a drug is administered that results in the enhancement of syndecan expression to levels sufficient to facilitate cellular differentiation in the degenerative stages of tissues. Such drugs are termed “syndecan-inducing agents.” Syndecan-inducing agents include growth factors and the derivatives of such factors that retain growth-factor activity. Examples of such growth factors include FGF-2, and TGF-β, whether administered separately or together.

Even more preferred is a syndecan-inducing agent that has good tissue and cell penetration so that it can directly interfere with suppressor(s) of syndecan expression within cell nuclei. Such a syndecan-inducing agent is the antitumor drug toremifene. When toremifene, known to have good plasma membrane penetration, is administered to the hormone-transformed epithelial cells with reduced syndecan expression, the cells reverse their lowered syndecan expression, and evidence a syndecan level close to that observed in normal, non-transformed cells (FIG. 8). This demonstrates that syndecan-inducing agents useful in the methods of the invention are known and available and that such agents can specifically prevent cells from becoming malignant by blocking suppression of syndecan expression. Another useful drug in this regard is tarnoxifen.

Syndecan-inducing agents may be administered using currently available preparations, or in any pharmaceutically acceptable vehicle. The route of administration may be any route that delivers efficacious levels of the drug to the desired active site, for example, by injection.

For parenteral administration, preparations containing one or more syndecan-inducing agents may be provided to the patient in need of such treatment in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose and the like.

The syndecan-inducing agent of the invention can be employed in dosage forms such as tablets, capsules, powder packets, or liquid solutions for oral administration provided that the biological activity of the syndecan-inducing agent is not destroyed by the digestive process and that the characteristics of the compound allow it to be absorbed across intestinal tissue. Syndecan-inducing agents may also be administered by means of pumps, or in sustained-release form. The syndecan-inducing agents used in the method of invention may also be delivered to specific organs in high concentration by means of suitably inserted catheters, or by providing such molecules as a part of a chimeric molecule (or complex) which is designed to target specific organs.

Administration in a sustained-release form is more convenient for the patient when repeated injections for prolonged periods of time are indicated.

The composition containing the syndecan-inducing agent can be manufactured in a manner which is in itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, lyophilizing or similar processes. The compositions of the present invention that provide the syndecan-inducing agent find utility in their ability to slow or prevent tumor growth or tumor reappearance. The syndecan-inducing compositions of the invention utilize the body's own mechanisms for promoting differentiation of specific cell types.

In intravenous dosage form, the compositions of the present invention have a sufficiently rapid onset of action to be useful in the acute management of tumor growth. Additionally, a low potency version is useful in the management of disorders wherein a tumor has been effectively treated and the patient appears to be in remission, but it is desired to maintain sufficient levels of syndecan-inducing agents in the patient so as to assist the body in preventing a recurrence of the tumor.

Typical doses of toremifene or tamoxifen, and other such syndecan-inducing agents useful in the methods of the invention for treatment of humans or other animals are 20-600 mg daily, and preferably 20-60 mg daily.

The Examples below are for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE I

Reversal of hormone-induced transformation by exogenous syndecan expression. As previously described (Leppä et al., Cell Regulation 2:1-11 (1991)), S115 mouse mammary tumor cells were routinely cultured in DMEM. For experimental studies involving hormone treatment, inactivated fetal calf serum (I-FCS) was replaced with 4% dextran charcoal-treated fetal calf serum (DCC-FCS), which eliminates endogenous steroids from serum, and used either with or without testosterone (10 nM) and with or without dexamethasone (10 nM or 1 μM). Cells were plated at a density of 10,000 cells/cm² and the medium was replenished every 3 days.

Plasmid pUC19-hsynpr7 containing human syndecan cDNA (Mali et al., J. Biol. Chem. 265:6884-6889 (1990)) was digested with NaeI restriction endonuclease, and the derived 336 bp long-fragment was separated in and eluted from low melting agarose gel. Plasmid pUC19-hsyn4 (Mali et al., J. Biol. Chem. 265:6884-6889 (1990)) was digested with NaeI and HindII (polylinker site), and the plasmid-containing fragment starting from base 487 was isolated. The NaeI fragment from hsynpr7 was ligated to the pUC-hsyn NaeI/HindII digested vector. The orientation of the insert was verified by restriction enzyme analysis and sequencing. The derived plasmid, containing the full coding region of human syndecan core protein, was named pUC19-hsynfull. This plasmid was further digested with BamHI and SphI (polylinker site). A fragment containing syndecan coding region bases 150-1461 was isolated and blunt-ended, using Klenow and T4 DNA polymerase. Finally, this fragment was ligated to SalI-linearized and blunt-ended pMAMneo vector (Clontech; Palo Alto, Calif.), resulting in a chimeric gene containing a RSV-MMTV-LTR promoter connected to the human syndecan coding region and to an SV-40 polyadenylation signal. The orientation was confirmed by restriction enzyme digestions. The plasmid was named pMAMneo-hsyn.

For control transfections, a 642 bp long-HindIII/PvuII fragment of the human growth hormone gene (consisting of exons 4 and 5; Bornstein et al., J. Biol. Chem. 263:1603-1606 (1988)) was blunt-ended and cloned into the same pMAMneo vector, as described above. This control construct was named pMAMneo-hGH.

All plasmids were isolated using the CsCl density gradient method. Before transfections, plasmids were linearized with MluI, chloroform/phenol extracted and ethanol precipitated.

Transfections were performed using Lipofectin™ (BRL), according to manufacturer's instructions. After selection for two weeks (G418; 750 μg/ml, Sigma), surviving clones were isolated from growth plates using cloning cylinders. The expression of human syndecan or growth hormone (consisting of exons 4 and 5) mRNAs was then confirmed by RNA isolation and Northern blot analysis. Clones expressing high levels of transfected genes were selected for further studies and characterizations. These stock cells were routinely cultured in the presence of G418 (300 μg/ml).

For the measurement of exogenous syndecan expression total RNA was isolated from wild-type S115 cells and cells transfected with human syndecan or growth hormone genes. RNA was extracted using 4M guanidine isothiocyanate and CsCl pelleting, as earlier described by Chirgwin et al., Biochemistry 18:5294-5299 (1979)). RNA from normal mouse mammary NMuMG and normal human mammary HBL-100 cells was used for comparison. RNA aliquots of 15 μg were separated in 1% formaldehyde agarose gels by electrophoresis and transferred to a GeneScreen Plus™ hybridization membrane (New England Nuclear). Blots were hybridized with multiprime (Amersham) labeled inserts of either mouse (PM-4) (Saunders et al., J. Cell Biol. 108:1547-1556 (1989)) or human syndecan (pUC19-hsyn4 BamHI 1.1 kb fragment) (Mali et al., J. Biol. Chem. 265:6884-6889 (1990)), or with human growth hormone exons 4 and 5 (hGH) (Leppä et al., Proc. Natl. Acad Sci. USA 89:932-936 (1992)) cDNAs, using the high stringency conditions suggested by the manufacturer of the membrane (New England Nuclear). All techniques based on modern molecular biology are fully explained in the literature such as in the laboratory manual entitled Current Protocols in Molecular Biology.

Anchorage independent cell growth was measured in a soft agar colony assay. The six well-plates were first covered with an agar layer consisting of 2 ml DMEM, 0.5% agar and 4% DCC-FCS. The middle layer contained 10⁴ cells in 0.5 ml DMEM supplemented with 0.33% agar and 4% DCC-FCS, with or without 10 nM testosterone. The uppermost layer, consisting of medium (2 ml), was added to prevent drying of the agarose gels. The plates were incubated at 37° C. in 5% CO₂ for 12 days after which cultures were evaluated and photographed.

Tumorigenicity of S115 wild type cells, one hGH transfected control clone and two clones expressing human syndecan-1 was measured in nude mice. Cells were cultured in DMEM containing 5% FCS and 10 nM testosterone. After four days in culture, cells were harvested with trypsin, washed, and 10⁷ cells suspended in 0.2 ml of DMEM were injected subcutaneously into the backs of athymic male nude mice (balb-C). A silastic testosterone capsule, which is known to increase the growth rate of S115 cells (King et al., J. Steroid. Biochem. 7:869-873 (1976)), was simultaneously implanted. Nude mice were examined regularly for tumor development and the size of the palpable tumors measured at intervals.

EXAMPLE II

Growth factors enhance syndecan expression. NMuMG mouse mammary epithelial cells and 3T3 (NIH) mouse fibroblasts were routinely cultured in bicarbonate-buffered Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10% FCS (GIBCO) and antibiotics, as previously described (Elenius et al., J. Biol. Chem. 265:17837-17843 (1990)). For experiments, cells were plated at equal density on culture dishes (Nunc) and grown to 60-70% confluency. Twenty-four hours before supplementing the medium with growth factor(s), the medium on the cells was replaced with fresh medium containing 2% CMS-FCS (Vogel et al., Proc. Natl. Acad. Sci. USA 75:2810-2814 (1978)). Equally treated cultures without growth factor addition served as negative controls. Porcine TGFβ1 (R&D), recombinant human bFGF (Boehringer) and murine EGF (Sigma) were used in final concentrations of 2.5 ng/ml (100 pM), 10 ng/ml (570 pM) and 1.2 ng/ml (200 pM) respectively, in all experiments. For quantitation and isolation of cell surface syndecan, media were discarded at time points indicated in the text and the cell layers were washed twice with ice cold phosphate buffered saline (PBS). Cells were scraped with a rubber policeman into ice cold PBS supplemented with 0.5 mM EDTA and centrifuged. After subsequent washes by resuspension and centrifugation the cell numbers were measured by counting the nuclei with a Coulter Counter (Coulter Electronics).

For quantitation of syndecan intercalated into the cell membrane, syndecan ectodomain was released by incubating washed cells in 20 μg/ml bovine pancreatic trypsin (Type III; Sigma) in PBS for 10 min on ice bath. After incubation the cells were centrifuged, leaving the ectodomain in the supernatant (Rapraeger et al., J. Biol. Chem. 260:11046-11052 (1985)). Sample volumes equal to 400,000 or 200,000 cells for 3T3 or NMuMG cells, respectively, were loaded onto a cationic nylon membrane (Zeta-Probe; BioRad) in a minifold-slot apparatus (Schleicher and Schuell), as previously described (Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Nonspecific binding was blocked by incubating the membrane for one hour at room temperature in PBS containing 10% FCS. Syndecan attached to the membrane was detected with a monoclonal antibody against mouse syndecan core protein (mAB 281-2) (Jalkanen et al., J. Cell Biol. 101:976-984 (1985)) that was radioiodinated by the chloramine-T oxidation method (Stähli et al., Meth. Enzymol. 92:242-253 (1983)). The membrane was incubated overnight at 4° C. with ¹²⁵I-labeled 281-2 in PBS+10% FCS (10,000 CPM/ml). After five washes with PBS the bound antibody was visualized by autoradiography.

The accumulation of syndecan ectodomain into the medium was estimated by taking samples corresponding to 1/50 (3T3 cells) or 1/100 (NMuMG cells) of the total volume of the remaining medium at selected time points. The samples were analyzed by loading them onto a nylon membrane as described above. The autoradiography signal was quantitated with a GelScan XL ultroscan densitometer (LKB) using GelScan XL 2400 software (LKB).

For syndecan purification, cells were radiolabeled for 24 hours in low sulfate DMEM (MgCl₂ substituted for MgSO₄; 2% CMS-FCS) with 100 μCi/ml ³⁵SO₄ (New England Nuclear) in the presence or absence of growth factor(s). Cell surface trypsin-releasable material was collected, as described above, and after dialysis against Tris-buffered saline (TBS), the sample was loaded onto a 281-2-Sepharose CL-4B immunoaffinity column (Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Bound material was eluted with 50 mM triethylamine (TEA) (pH 11.5) and the amount of radioactive PG in each fraction was analyzed using cetylpyridiumchloride-impregnated Whatrnan 3MM filter discs (Rapraeger et al., J. Biol. Chem. 260:11046-11052 (1985)). For interaction experiments, fractions containing most of the labeled PG were pooled and dialyzed against PBS.

To obtain unlabeled syndecan ectodomain for interaction assays (see below) the same procedure was used except that no radioactive sulfate was added to the culture medium and the syndecan containing fractions eluted from the immunoaffinity column were detected by immuno-dot assay using mAB 281-2. The estimation of the molar concentration of syndecan was based on the use of previously determined syndecan concentration by total amino acid analysis (Jalkanen et al., J. Cell Biol. 106:953-962 (1988)).

SDS-PAGE and Western Blot—For western blot experiments, cells were cultured 24 hours with or without growth factor(s). Syndecan ectodomain containing material released from the cell surface by trypsin treatment was fractionated on SDS-PAGE gradient (2-15%) gel (O'Farrel, J. Biol. Chem. 250:4007-4021 (1975)). After electrophoresis, samples were transferred onto a Zeta-Probe membrane by electroblotting with a 2005 Transphor apparatus (LKB). The syndecan antigen on the filter was detected with radioiodinated mAB 281-2 and the filter was washed, as described above for slot blot analysis.

Northern Blot—RNA was isolated from 3T3 and NMuMG cells using 4 M guanidine isothiocyanate and CsCl density centrifugation (Chirgwin et al., Biochemistry 18:5294-5299 (1979)). RNA samples were size-separated on a 1% agarose formaldehyde gel, transferred to a GeneScreen Plus™ membrane (New England Nuclear) and hybridized with a multi-prime (Amersham) labeled partial cDNA clone for mouse syndecan (PM-4) (Saunders et al., J. Cell Biol. 108:1547-1556 (1989)). After hybridization, the membrane was washed in 2×SSC and 1.0% SDS at 65° C. (high stringency conditions). For rehybridization with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Fort et al., Nucleic Acid Res. 13:1431-1442 (1985)), the bound PM-4 probe was removed as recommended by the manufacturer of the filter (NEN).

EXAMPLE III

Induction of syndecan mRNA expression in human breast cancer cells (MCF-7) growth-inhibited with toremifene. The steroid-responsive human breast cancer cell line MCF-7 was used to study the expression of human syndecan under different growth conditions regulated by estrogen and anti-estrogen. Cells were plated at a density of 1.2×10⁶ cells/100 mm of plastic culture dish and grown as monolayer cultures in 10 ml per dish of phenol red-free DMEM medium with 5% dextran/charcoal treated fetal calf serum (DS-FCS), 2 mM L-glutamine and 3 μg/ml insulin. For hormone-treatment, 1 nM of 17β-O-estradiol (E ₂), alone or with 1-6.25 μM toremifene, dissolved in 70% ethanol, was added to the culture medium on the day following plating. The cells were cultured for 6 days, and the media were changed every second day. For RNA extraction, cells were washed in situ with PBS and scraped from the plates in 4 M guanidine isothiocyanate.

EXAMPLE IV

Treatment of Steroid-Responsive Tumors in Patients. Patients diagnosed as having a steroid-responsive tumor selected from a breast tumor, an endometrium tumor, a prostate gland tumor or a mesenchymal tissue tumor are administered a composition that contains efficacious amounts of the anti-steroid agent toremifene or tamoxifen, or efficacious amounts of the growth factor bFGF, TGF-β or bFGF together with TGF-β, in amounts ranging from 20-600 mg per day, depending upon the extent of the tumor, the patient's age, the patient's sex, and other treatments such as are taken into consideration in designing such chemotherapeutic protocols. The syndecan-inducing agent is administered for a period of time sufficient to increase syndecan levels in the tumor cells, such that the tumor cells now assume a more differentiated phenotype and such that the growth of the tumor is arrested or significantly slowed by the treatment.

EXAMPLE V

Stimulation of Hair Growth in Epidermal Skin Cells. Patients diagnosed as being in need of increased hair growth in the scalp region are administered a composition that contains efficacious amounts of the anti-steroid agent toremifene or tamoxifen, or efficacious amounts of the growth factor bFGF, TGF-β or bFGF together with TGF-β, in amounts ranging from 20-600 mg per day, depending upon the extent of the needed hair growth, the patient's age, the patient's sex, and other treatments such as are taken into consideration in designing such protocols. The syndecan-inducing agent is administered for a period of time sufficient to increase syndecan levels in the epidermal cells, such that hair growth is significantly increased by the treatment.

EXAMPLE VI

Determination of Mouse Syndecan Promoter and Enhancer Activities. The mouse syndecan-1 gene has been cloned and characterized up to −10 kbs upstream from the transcription start site. To determine the specific activities of different proximal promoter regions (up to −2 kbs from the start site) and enhancer regions (from −2 to −10 kbs) we have made plasmid constructs in which these regions were cloned into pCAT basic or pCAT promoter vectors, containing the CAT reporter gene. The reporter CAT gene produces the enzyme chloramphenicol acetyltransferase, which transfers the n-butyryl moiety of n-butyryl CoA to chloramphenicol. The n-butyryl chloramphenicol can be separated from native chloramphenicol by extraction with xylene.

For the proximal promoter, a deletion series was made (HindIII, HindII, BglII, StuI, DraI, ClaI, BamHI and PstI-XhoI) and the resulting fragments were cloned into the pCAT basic vector. For enhancer areas, three XbaI fragments were cloned into a pCAT promoter vector, where the SV 40 promoter was displaced by the BglII-XhoI fragment from the syndecan promoter.

The plasmid constructs were transiently transfected into eukaryotic cells by calcium phosphate precipitation simultaneously with a β-Galactosidase expressing vector to determine transfection efficiency. After a four hour incubation, cells were treated with 15% glycerol and grown for approximately 48 h in cell culture medium. Cells were then scraped from dishes in TEN-buffer and the cytoplasmic extract was obtained by repeated freezing and thawing. β-Galactosidase activity was obtained in the cytoplasmic extract by adding 0.1 M sodium phosphate, 45 mM mercaptoethanol and 0.2 mg O-nitrophenyl-s-galactopyranoside (ONGP). This was incubated from 2 hours to overnight and the color reaction was measured spectrophotometrically at 420 nm.

CAT activity was determined by adding 0.25 M Tris buffer, 25 ng n-butyryl CoA and 0.0626 μCi of ¹⁴C-chloramphenicol to the cytoplasmic extract. Samples were incubated overnight, extracted with xylene and the radioactivity present was measured by scintillation counting. CAT activity was corrected for transfection efficiency as determined by assays of β-galactosidase activity.

The cells used for proximal promoter constructs were 3T3 NIH, S115 (either hormone-treated or not) and nMuMG cells. For enhancer constructs we used 3T3 NIH cells grown in 2% CMS medium and MCA 3D cells grown in Ham's medium. 3T3 NIH cells were also used to test the effect of growth factors in 2% CMS medium with 10 ng/ml FGF-2 and 2ng/ml TGFβ-1. MCA 3D cells were used to test the effect of 10 ng/ml KGF.

By measuring the CAT activity present in transfected cells, the effect of inserted syndecan gene regions on gene transcription was determined. Results indicated that both a suppressor element (FIGS. 9a-9 b) and an enhancer element (FIG. 10) are upstream of the syndecan transcription initiation site. The suppressor element is located between 250 and 600 base pairs upstream from the transcription initiation site. The enhancer element is located between 8,600 and 9,300 base pairs upstream from the syndecan transcription initiation site.

EXAMPLE VII

Activation of Syndecan-1 Gene Transcription FGF-2 in NIH3T3 Cells. Syndecan-1 expression is usually very low in mesenchymal cells if compared to epithelial cells. However, syndecan-1 expression can be transiently induced in many mesenchymes (Vainio et al., Devel. Biol. 134:382-391 (1989); Vainio et al., Devel. Biol. Dev. Dyn. 194:105-117 (1992)). Syndecan-1 expression is upregulated in 3T3 cells after 24 hr simultaneous FGF-2 and TGF-β exposure (See Elenius et al., J. Cell Biol. 114:585-595 (1991)).

To demonstrate that FGF-2 could alone activate the syndecan-1 gene, 3T3NIH cells were exposed to 10 ng/ml of FGF-2 for 0, 4, 6, 8, 12, 24 and 28 hrs to test the activation of the endogenous syndecan-1 gene. Following incubation with FGF-2, syndecan MRNA was isolated and quantified. Control incubations without FGF-2 were also performed. Syndecan mRNA was isolated and quantified without FGF-2 treatment following 6, 12, and 24 hr incubations. The quantification of syndecan mRNA production can be seen in FIG. 11a. GAPDH mRNA was used as a loading control. Based upon these data, syndecan expression is significantly enhanced (several fold) after 4 hrs of incubation with FGF-2. The enhancement appears to be maximal at about 6 hrs of FGF-2 incubation, and decreases thereafter. In the Figure, two individual exposures for each time point are presented.

Nuclear run-off experiments revealed that this upregulation was transcriptional. The same cells were exposed to 10 ng/ml of FGF-2 for 4 hours and 24 hours with or without exposure to FGF (Control) followed by the isolation of nuclei for the run-off experiment. c-jun or nur transcription was used as a positive control and β-actin as a loading control. In nuclei isolated 4 hours after FGF exposure the level of transcription of the syndecan-1 gene was elevated as were the levels for c-jun and nur as positive controls. The transcription of syndecan-1 was not detectable after 24 hours (FIG. 11d), in agreement with the results of Northern hybridization.

This result suggests that the syndecan-1 gene is a direct target for an FGF-2 induced signal. Moreover, this targeting can be modulated by signals produced by other growth factors, like TGF-β, which alone cannot activate the syndecan-1 gene.

EXAMPLE VIII

FGF-induced Transcription of the Syndecan-1 Gene is Based on the Activation of a Far Upstream Enhancer. In order to characterize the transcriptional elements responsible for syndecan-1 expression following FGF-2 exposure, the 5′-region of the gene was cloned and sequenced 12 kb upstream from the TATA box.

The mouse syndecan gene was previously cloned and sequenced to −9.4 kb (See, supra; Vihinen et al., J. Biol. Chem. 271:12532-12541 (1996)). To sequence further upstream an XbaI fragment (Xb6) from the cosmid clone was subcloned into pBluescript KS M13 (+/−) vectors (Stratagene). DNA sequencing was performed by the dideoxy chain termination method. Sequence data base comparisons were made with the Wisconsin package (Genetics Computer Group, Inc.) and the Transcription Factor Database (TFD). The sequence of Xb6 has been deposited at the EMBL Sequence Data Bank under accession number Z 22532 (SEQ ID No. 3).

Various constructs were made containing different fragments of the 11.5 kb syndecan upstream regulatory region together with the 1.1 kb segment containing the proximal promoter of the syndecan-1 gene. Plasmid pXb6CAT contains an XbaI fragment from −9.4 kb to −11.6 kb operably linked to the proximal promoter sequence, which in turn is operably linked to the CAT gene. Plasmid pXb4CAT contains an XbaI restriction fragment from −5.0 to −9.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid pXb2/5′CAT contains anXbaI fragment from −2.4 kb to −4.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid p-2.5 CAT contains a HindIII-XhoI fragment from −1.3 kb to −2.4 kb linked to the segment containing the proximal promoter, which is operably linked to the CAT gene. Plasmid pSyn Prom CAT contains no enhancer segment, but contains the proximal promoter segment operably linked to the CAT gene. The structure of these plasmids is shown in FIG. 11b. In each of these constructs the distance between the potential enhancer activity and the Syn Pro segment is conserved; about 2.5 kb separates the fragments containing the potential enhancer from the SynPro segment.

Each of these plasmids was transiently transfected into 3T3NIH cells, using the calcium phosphate precipitation method (Chen and Okayama, Mol. Cell Biol. 7:2745-2752 (1987)) as follows. For transient transfections NIH3T3 cells were plated at equal density on six-well plates (Falcon) two days before transfection. Plasmid DNA was transfected into cells by calcium phosphate method (Chen and Okayama, Mol. Cell Biol. 7:2745-2752 (1987)). A β-galactosidase expressing plasmid (pSV-β-galactosidase, Promega) was co-transfected with CAT constructs to monitor transfection efficiencies. Three parallel transfections were used in every assay. Directly after transfection growth factors were added, media change the next day and cells were harvested after two days. CAT activities were measured by liquid scintillation and β-galactosidase activities spectrophotometrically at 420 nm as described by Vihinen et al., J. Biol. Chem 268:17261-17269 (1993)). As a control, other samples of the cells transfected with each plasmid were treated with buffer without FGF-2 for the same time periods. Only plasmid pXb6 responded to FGF-2 treatment with an increase in CAT gene expression. See FIG. 11b. The cells transiently transfected with the other plasmids showed no significant difference in CAT gene expression with FGF-2 treatment and without FGF-2 treatment. These results indicate that enhancer activity is localized to within the region between −9.4 kb to −11.6 kb upstream of syndecan transcription initiation site.

This region of the syndecan upstream regulatory region was further cleaved into smaller fragments and assayed similarly. See FIG. 11c. Specifically, plasmid pXSp2 contains a 1.14 kb XbaI-SphI restriction fragment from −11.6 kb to −10.5 kb. Plasmid pXSp1 contains a 1.05 kb SphI-XbaI restriction fragment from −10.5 kb to −9.4 kb. Plasmid pXS2 contains a 0.36 kb SphI-EarII fragment from −10.5 kb to −10.1 kb. pXB3 comprises a 0.30 kb EarI-EarI restriction fragment segment of the enhancer region from −10.1 kb to −9.8 kb of pXB6. pXX1 is a 0.39 kb EarI-XbaI restriction fragment of the enhancer region from −9.8 kb to −9.4 kb of pXb6. pFiRE comprises a 0.28 kb PstI-StyI restriction fragment of the enhancer region from −10.3 kb to −10.0 of pXb6. pFiRE_(REV) contains the fragment of enhancer region from pXb6 inserted in the opposite orientation.

For the gene expression analysis the fragments of the syndecan-1 upstream region were subcloned from mouse genomic clones into the XbaI site of the pCATProm vector in which a mouse syndecan-1 promoter region (−1310 to +140) was fused to the CAT reporter gene (Vihinen et al., J. Biol. Chem. 271:12532-12541 (1996)). The pX-HIIICAT plasmid was prepared by cloning HindIII-XhoI promoter fragment (−2400 to +140 bp) into the promoterless pCAT basic vector (Promega). For pXSp1 and 2 plasmids, SphI fragments −11.4 to −10.5 kb and −10.5 to −9.4 kb, respectively) were deleted from pXb6CAT (−11.6 to −9.4 kb). Constructs pXS2, pXB3 and pXX1 were generated by ligating SphI-EarI/blunt (−10.5 to −10.1 kb), EarI/blunt fragment (−10.1 to −9.8 kb) and EarI/blunt-XbaI (−9.8 to −9.4 kb) from pXSpI into SphI-AccI/blunt, AccI/blunt or XbaI-SphIlblunt sites, respectively, into the pCATProm vector. Blunt ended fragments were made by T4 polymerase (Promega). For FiRE and FiRE_(REV) a PstI-StyI/blunt fragment was subcloned into PstI-EcoRV or PstI-SmaI sites of the pBluescript vector and then transfected to Xba-SphI sites in pCATProm vector. These constructs were then used to transient transfect NIH3T3 cells as described supra, and CAT activity was measured.

The 280 base pair element inserted into pFiRE retained full FGF-2 dependent enhancement of CAT expression. See FIG. 11c. This enhancement was also maintained when the 0.28 kb fragment was inserted in the opposite orientation (pFiRE_(REV)). In addition, plasmid pXSp1, which encompasses the sequence of pFiRE, also exhibited a FGF-2 dependent enhancement in CAT expression. These results localize the enhancer element to a 280 bp region from nucleotide −10,280 to nucleotide −10,000 with the first nucleotide at the translation initiation site numbered as +1.

EXAMPLE IX

Effect of Various Growth Factors on Enhancer Activation in NIH3T3 Cells. Plasmid pFiRE was transfected into 3T3NIH cells as described, supra. The cells were starved in DMEM medium supplemented with 5% fetal calf serum to about 70% confluence. Fetal calf serum was then replaced with 2% carboxy-methyl-Sephadex eluted fetal calf serum (CMS) 12 hrs before adding the growth factors. Various growth factors in the same medium, 5% fetal calf serum (FCS) supplemented medium or a control containing the medium, without any added growth factors were incubated with the transfected cells for 2 days, and CAT activity was measured. The effect of 10 ng/ml of FGF-2, FGF-1, FGF-4, FGF-7 (KGF), EGF, IGF1, TGFα, IFNγ, and PDGF and 2 ng/ml TGF-β were tested. The human recombinant growth factors were purchased from Peprotech (FGF-1, -2, and -7), Boehringer (FGF-2) or from Calbiochem (PDGF) TGF-β was purchased from Sigma. The effect of the added growth factors and FCS on DNA synthesis was also tested by a 125-IdU incorporation assay. This cell proliferation assay was carried out by incubating cells for 4-6 hrs with 0.25 μCi/ml 5-[¹²⁵I]iodo-2′-deoxyuridine (IdU, Amersham), washed several times with PBS and solubilized in 1 M NaOH. Radioactivity was measured using a gamma counter (Wallac).

The results of this experiment are reported in FIGS. 12a-12 c. The results indicate that all of the tested growth factors significantly increase the level of incorporation of 125IdU above that of the control, indicating that the growth factors were taken up by the transfected 3T3 NIH cells and were functional (FIG. 12b). Although all of the growth factors known to act on 3T3 cells stimulated 3T3 proliferation, FGFs were the only growth factors to clearly increase reporter gene activity (FIG. 12a). FGF-1 and FGF-4 had a lower effect those FGF-2 (FIG. 12A). FGF-7, known to act only on epithelial cells, had no effect. Some FGF-1 preparations, from the different commercial sources, lacked the ability to activate FiRE, while still inducing cell proliferation. Only FGF-2 revealed clear enhancer activation. However, 5% FCS slightly stimulated the enhancer.

In addition, 5% FCS only induced a small response despite containing several growth factors. PDGF, IGF and EGF enhanced cell proliferation to a similar extent as FGF-2, but had no effect on enhancer activity. Thus, stimulation of different tyrosine kinase receptors does not lead to activation of FiRE, nor does proliferation alone correlate with activation of FiRE. Consequently, FiRE cannot be regarded as a simple proliferation related target of growth factors.

As the original construct contained over 1 kb of syndecan-1 proximal promoter, we wanted to rule out the possible suppressive action of this basal promoter on the function of the other growth factors. Therefore, nearly all of the promoter was deleted, leaving only 98 bp of the proximal promoter (p-271CAT), which included only the putative TATA-box without any upstream regulatory elements (Vihinen et al., 1996). As shown in FIG. 12c the removal of the syndecan promoter had no effect on the pattern of growth factor induced FiRE activation, indicating that the growth factor specificity is not regulated by the proximal promoter.

EXAMPLE X

The Enhancer Comprises Five DNA Binding Motifs. Plasmid pFiRE was end-labeled using Klenow fragment and α²²PdCTP. DNAse I footprinting was performed with labeled pFiRE vector alone (naked), together with FGF-induced nuclear extracts (FGF-2), and together with non-induced nuclear extracts (control). To obtain nuclear extracts, NIH3T3 cells were plated on 16 cm dishes and treated with or without FGF-2 for 1 day. Nuclear proteins were extracted using a modification described by Lee et al., Gene Anal. Techn. 5:22-31 (1988). Protein concentrations were measured by the Bradford method.

For footprinting, pBluescript containing pFiRE was cut with HindIII, end-labeled with [α³²P]dCTP using Klenow DNA polymerase (Promega) and digested with XbaI. The labeled and PAGE purified DNA was incubated for 10 minutes at room temperature with about 40 mg of crude nuclear extract, 2 μg of poly-3[I-C] (Boehringer Mannheim) in a reaction buffer (10 mM TrisCl, pH 8; 5 mM MgCl₂, 1 mM CaCl₂, 2 mM DTT, 50 mg/ml BSA and 100 mM KCl) containing 0.1 or 1 unit of DNAseI (Boehringer Mannheim). The reaction was stopped after 2 minutes. A chemical G+A sequencing ladder (Maxam & Gilbert, Meth. Enzymol. 65:499-560 (1980)) was run along with the digestion products on a 6% sequencing gel. The results are shown in FIG. 13a. Five footprinted motifs ranging from 14 to 38 bp in length in close proximity to each other and covering a total of 170 bp were revealed and are marked with nwnbered boxes. Binding of nuclear factors to motifs 3, 4 and 5 appear to be FGF-2 dependent, whereas binding to motifs 1 and 2 appear to be FGF-2 independent. The A+G sequence of pFiRE was run alongside the footprinting ladders.

EXAMPLE XI

Sequence of the FGF-2 Inducible Enhancer. The 280 bp fragment of pFiRE containing the syndecan enhancer was sequenced. This sequence is depicted in FIG. 13b. The footprinted motifs observed in Example X are underlined. It was determined that these footprinted motifs together restore 50-70% of the original enhancer activity. The sequence of motif 3, which contains the binding site for FGF-inducible nuclear factor FIN-1, was compared to consensus binding sites for transcription factors. No significant homology between this motif and the consensus sequences was observed.

EXAMPLE XI

The Enhancer is Bound by FGF-2 Inducible and Non-inducible Nuclear Factors. Gel retardation analysis for each enhancer motif (1-6) was performed with double-stranded oligonuceotides.

The double-stranded oligonucleotides were end-labeled with [α-32P]dATP (ICN Biomedicals) by the polynucleotide kinase (Promega). The sequence of these oligonucleotides is:

Motif Sequence 1 5′-dGCTGGCACAC CCACCGTCAC GAGAGCTTCC-3′ SEQ ID NO: 6 2 5′-TTGGCACACC TGGGAGGATG-3′ SEQ ID NO: 7 3 5′ AGTGGTTCAG GGTGACTCT-3′ SEQ ID NO: 8 4 5′-AGGAGTGAGC CATGCCACC-3′ SEQ ID NO: 9 5 5′-CTGGGTCATT GATGACTG-3′ SEQ ID NO: 10

In a 12 μl reaction, 5 μg of the nuclear extracts were incubated with labeled oligonucleotide, 2 μg of poly-d[I-C] and 2×reaction buffer, i.e., 30 min Tris, pH 7.5; 100 mM NaCl; 2 mM EDTA; and 10% glycerol for 15 minutes at room temperature. Non-labeled competitor oligonucleotides were used at 100-fold molar excess. The complexes were analyzed by electrophoresis in a 4.5% polyacrylamide gel. For supershifts, 0.1 μg of specific antibody was added to the reaction 15 minutes before the labeled oligonucleotide.

Each motif element was treated with nuclear extracts derived from FGF-2 treated (f or FGF) or untreated (C or control) 3T3 cells. An SPI consensus oligo was used as a control to test the functionality of the nuclear extracts. The results are given in FIG. 14a. Binding of at least two protein complexes for each motif were observed. The results indicate that enhancer motifs 3, 4 and 5 bind to a factor present in nuclear extracts from FGF-2 treated 3T3 cells. However, binding of enhanced proteins to motifs 1 and 2 were evident in stable FGF-2 stimulated and nonstimulated 3T3 cells.

These motifs was further analyzed by competing the binding of nuclear factors with a molar excess of specific (s) and non-specific (ns) oligonucleotides. The non-specific oligonucleotide for motifs 3, 4 and 5 was the motif-1 sequence, while the non-specific oligonucleotide for motifs 1 and 2 was the motif 3 sequence. The resulting complexes were analyzed by the gel retardation analysis described above. The results are depicted in FIGS. 14b (motif 1), 14 c (motif 2), 14 d (motif 3), 14 e (motif 4) and 14 f (motif 5). Specific binding is indicated by an arrow. The result indicated that all motifs shifted at least one specific band in the gel retardation assay. The results also show that motifs 3, 4, and 5 bind at least one specific FGF-2 inducible nuclear protein. Motifs 1 and 2 do not show binding to an FGF-2 inducible factor. From these results it can be concluded that all 5 motifs are in use. Motifs 1 and 2 are occupied constitutively by nuclear proteins, while motifs 3, 4, and 5 are occupied only after FGF exposure of 3T3 cells.

EXAMPLE XIII

Deletion Mutants of FiRE. To determine whether all binding sites are required for enhanced activation, deletion mutants for each motif were generated by PCR. For motif 1, the 3′ end of FiRE was deleted by generating a PCR product ranging from the 5′ end of FiRE to motif 2. For motif 2, the E-Box was replaced by a KpnI restriction site. For motifs 3 and 4, a central 10 bp sequence was replaced with a 10 bp SpeI restriction site. For motif 5, a PCR product from motif 4 to motif 1 was generated. The mutant DNA was ligated into a CAT reporter plasmid as described supra. Cells were treated with FGF-2 and CAT assays were performed as described. The results of this experiment are depicted in FIG. 21. The modification of motifs 1, 3, 4 and 5 resulted in the loss of a significant reduction of FiRE transcriptase activation. However, the modification of motif 2 has little or no effect on transcriptional activation. This result supports the involvement of 4 of the 5 motifs in the activation of FiRE by FGF. Furthermore, it shows that at least two fos-jun complexes (AP-1) are required, but alone they are not sufficient to activate FGF induced transcription.

To determine whether the different inducible components of FiRE are due to direct post-translational modifications or whether they are newly synthesized, simultaneous cycloheximide and FGF treatment and subsequent protein extraction were performed. As shown in FIGS. 22a-22 d, the FGF-induced binding of AP-1s to motifs 4 and 5 as well as the binding of FIN-1 to motif 3 was blocked by cycloheximide. This indicates that all inducible proteins involved in FiRE require de novo protein synthesis, and that FiRE represents a secondary response element in FGF initiated signaling.

EXAMPLE XIV

Characterization of FiRE Binding Nuclear Proteins. The nucleotide sequences of FiRE (FIG. 13b) was compared to known sequences of different transcription factors stored in the Transcription Factor Database (TFD Sites) (Nisse). Motif 2 showed significant homology to a known transcription factor binding consensus site: an E-box. Motif 4 contained an AP-1-like consensus binding site with one mismatch, and motif 5 has two AP-1-like consensus sites. Motifs 1 and 3 do not contain known consensus sequences for transcription factors.

Various commercial consensus oligonucleotides, including AP-1, AP-2, AP-3, Ets, GATA, SIE, CRE, MEF-1, MEF-2, Max-Myc, SP-1, NFKB, and cEBP were used to test for competition with the nuclear factor-enhanced binding in order to reveal possible protein binding outside the established consensus sequence. The result of this experiment is reported in FIGS. 15a-15 e. A Max-Myc consensus oligonucleotide was able to abolish binding of nuclear factors to motif 2. (FIG. 15d). AP-1 consensus oligos were able to compete binding of nuclear factors to motifs 4 and 5. (FIGS. 15a and 15 b). Binding of nuclear factors to motif 3 were unexpectedly abolished by an AP-2 oligonucleotide even though there is no AP-2 consensus site on motif 3. (FIG. 15c). Binding of nuclear factors to motif 1 could not be competed with any of the consensus oligonucleotides tested. These results indicate that motifs 4 and 5 bind an AP-1 complex, motif 3 binds on AP-2 complex, motif 2 binds a basic helix-loop-helix (bHLH) factor such as myc/mad, and motif 1 binds an unknown nuclear factor.

In order to further confirm the results obtained from the competition experiments, the effect of adding specific antibodies to the gel retardation reactions was tested. Antibodies to c-fos and c-jun were able to remove specific binding to motifs 4 and 5 and to produce supershifts. Anti-USF or anti-ATF-3 antibodies analyzed at the same time, had no effect. Thus, the nuclear factors that bind to motifs 4 and 5 are AP-1 fos and jun proteins.

The effect of an AP-2 on binding to motif 3 was tested. While the AP-2 consensus oligonucleotide was able to abolish binding to motif 3, the AP-2 antibody had no effect (FIG. 15c). The ability of this antibody to bind antigen was tested with labeled AP-2 oligonucleotide using FGF-2 induced 3T3 cell nuclear extracts. It produced a supershift, showing that it was functional. Thus, the protein that binds motif 3 is an AP-2 related transcription factor, which is distinct from AP-2. This protein has been named FIN-1 for FGF inducible nuclear factor.

For motif 2, neither factor Max nor Myc antibodies had obliterating or supershifting effects. However, another bHLH protein, USF, is known to be constitutively expressed in 3T3 cells (Millerberger et al., Cell Biol. 5(5):2527-2535 (1995)). USF was shown to occupy motif2 on USF antibody removed the bond specific for binding to this motif without affecting the binding to other motifs (FIG. 15d).

EXAMPLE XV

Mass Analysis of the Enhancer Binding Nuclear Factors. A gel retardation experiment as described in Example XII was carried out, and the gel containing the oligonucleotides bound to proteins was exposed to UV light to crosslink the protein to the oligonucleotide. Specifically, the gel mobility assays were run as described supra. After the gel was run, it was exposed to 245 nm UV-light (3600J/em²) in a Strategene crosslinker. The gel was exposed for several hours, the specific bands were cut out, eluted overnight at 4° C., precipitated with ethanol, resuspended in Laemmli buffer, denatured at 95° C. for 5 minutes, and loaded onto a 10% SDS-PAGE together with a ¹⁴C-labeled molecular weight markers to analyze their molecular weights. The SDS-PAGE gel is shown in FIG. 15c, with the position of the molecular weight markers shown at the left. Lanes 1-5 correspond to motifs 1-5, respectively. The molecular weights of the nuclear factors were estimated after subtracting the mass of the oligonucleotide from the complex mass as indicated below:

MW Oligo + Motif Factor (FIG. 17) MW Oligo MW Factor 1 66 kDa 20 kDa 46 kDa 3 62 kDa; 90 kDa 12 kDa 50 kDa; 78 kDa

This experiment shows a reproducible 46 kDa band for motif 1 and two bands, 78 kDa and 50 kDa, for motif 3. These values have a margin of error of about ±3 kDa.

EXAMPLE XVI

The Enhancer is Not Functional in 3T3 Cells Expressing High Levels of Syndecan-1. Syndecan-1 transfected 3T3 cells (3T3 1.5) and wild type 3T3 cells were transiently transfected with the pFiRE construct, treated with FGF or a negative control, and analyzed for reporter gene (CAT) expression. The results are depicted in FIG. 16. The closed columns indicate relative CAT activity in control cells (without FGF treatment) while the labeled columns depict relative CAT activity in FGF-treated cells. The results indicate that activation of CAT expression by FGF-2 is significantly reduced in 3T3 1.5 cells which express high levels of syndecan.

EXAMPLE XVII

Targeting a Reporter Gene (lacZ) to Healing Wounds of Mouse Skin Using the FiRE Enhancer. To demonstrate the utility of the FiRE enhancer in targeting a desired gene product to sites of healing wounds, transgenic mice harboring the enhancer operably linked to the syndecan-1 proximal promoter and a reporter gene (lacZ) were produced. Two separate plasmid constructs were made. The first construct contained a 2.25 kb (XhoI-StuI fragment) of the syndecan-1 basic promoter region linked to β-galactosidase reporter gene (PTRG Syn Prom). This plasmid is depicted in FIG. 17. The second plasmid also contained an additional 280 bp (PstI-StyI fragment) encoding the enhancer element operably linked to the basic syndecan promoter (PTRG ENH). This plasmid is depicted in FIG. 18. Constructs were released from vector sequences by Not digestion; separated in agarose gels, and electroeluted. The constructs were then dissolved in injection buffer (10 mM Tris-HCl, pH 7.5 and 0.2 mM EDTA) dialyzed extensively for 3 days against the same buffer and the concentration was adjusted to 2 μg/μl.

Transgenic mice were generated by microinjecting (C57B1/6*DBA/2) fertilized eggs using standard methods (Hogan et al., supra). Founder animals were identified based on PCR analysis of tail DNA (Honley and Merle, Biotechniques 10:56 (1991)). Transgenic mouse lines were generated by mating founder animals to (C57B1/6*DBA/2) F1 males or females.

The Syn Prom insert resulted in 6 DNA positive and the ENH insert in 12 positive founder animals. DNA positive offspring were produced for further analysis. This analysis was targeted to wounded skin, where syndecan-1 expression is unregulated. Wounds were made to the mice tails and the wounded tissue was cut off 2 days later and stained with X-Gal. All 7 mice with the Syn Prom construct, without FiRE, showed no positive staining. However, all of the 12 FiRE containing mice were found to have staining at the wound sites. See FIGS. 19b-19 f. The intact skin did not show specific staining for either Syn Prom or FiRE. The most intense staining was seen in the wound edge keratinocytes.

Further away from the incision site the staining rapidly faded. The staining was first seen at the edge of the incision site on the sheet of keratinocytes starting migration toward the bottom of the wound. The leading edge of the migratory, keratinocytes was also heavily stained closer to the end of the incision. Where the keratinocytes that reached the bottom under the clot were stratifying, the staining was still robust. In the keratinocytes surrounding the hair follicles close to the wound, which are putative cells for a migration start point, revealed strong staining. This portion of staining corresponds with the proposed target cells of FGF-2 and/or FGF-7. However, FGF-7 is a stronger inducer of migration of keratinocytes and is the more probable inducer of FiRE in those cells. The pattern is superimposable with the syndecan-1 expression pattern. For example, in situ hybridization and antibody staining detect proliferating and migrating keratinocytes in stratifying epithelium as well as hair follicle keratinocytes close to the wound site. (Elenius et al. J. Biol. Chem. 267: 6435-6441 (1992)). This example can be extrapolated to other gene products, which have replaced the lacZ-gene to target expression of these genes to wound sites as a therapeutic means.

FiRE is Induced Selectively by FGF-7 in Keratinocytes. The ability of FiRE to enhance transcription was tested in the mouse keratinocyte cell line MCA3D (Kuliz-Martin et al., Carcinogenesis 4:1367-1377 (1983)). MCA3D cells were transfected as described supra for mouse NIH3T3 cells. Following transfection, the cells were treated with fetal calf serum, PDGF, FGF-1, FGF-2, and FGF-7 as described supra for the NIH3T3 cells. Surprisingly, FGF-2 only slightly activated FiRE although it stimulated cell proliferation. Serum, PDGF, and FGF-1 did not activate FiRE. However, FGF-7 strongly activated FiRE in these cells. Moreover, syndecan-1 mRNA expression is strongly induced by FGF-7. This shows that FiRE can be selectively activated by different members of the FGF family in different cell types.

The Syn Prom construct showed no activation with any growth factors when transfected into MCA3D mouse keratinocytes. In another epithelial cell line derived from mouse mammary gland epithelium (NMuMg), which is negative for FGFRs, none of the tested growth factors had an effect on the FiRE construct. This indicates that FIRE is both cell specific and activated through FGFRs.

EXAMPLE XVIII

Agents Activating AP-1 Are Not Sufficient for FiRE Activation. Since FiRE contains AP-1 transcription factors, whose activation is well characterized, we tested chemicals known to cause AP-1 dependent gene activation. These included the protein kinase C (PKC) activator 12-O-tetradecanoylphorpol-13-acetate (TPA), protein-phosphate-1 and -2 inhibitors okadaic acid and calyculin A, cAMP activator forskolin and tyrosine kinase phosphatase inhibitor orthovanadate (FIG. 24a). Inhibition of the tyrosine kinase phosphatase, causing continuous activation of tyrosine kinases, resulted in activation of FiRE. This suggests, that all the nuclear factors bound to FiRE could be activated by tyrosine kinase activation. Surprisingly however, none of the other compounds known to induce immediate early genes fos and jun and AP-1 driven promoters, were able to activate FiRE. This suggests that more than simple activation of the pathways elicited by these agents, e.g., PKC or cAMP dependent pathways, is required for the response on FiRE, induced by FGF.

All documents, e.g., scientific articles, cited herein are fully incorporated herein by reference. The contents of U.S. application Ser. No. 08/206,186, filed Mar. 7, 1994 and of U.S. application Ser. No. 07/988,247, filed Dec. 1, 1992 are expressly incorporated herein by reference. Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

14 26700 base pairs nucleic acid double linear DNA (genomic) CDS join(4378..4443, 22026..22106, 23001..23483, 23905..24039, 24251..24418) 1 TCTAGATATT CAAACTCACC AGATGGAGTG ATGTCCACCC CTATTGGTGG GAGTGACTAG 60 TCTTTCCTCT GTCTTCTGAC TCAGATGCTT AGCTAGCTCT TTAGGACCCA CCCTCACACC 120 TGCAAATAAT ACTTTATTTG CTCTCTTAGT ACCTTTAACC CAGTGGAGTT GACATGAGAA 180 ATTAACTACC ATAATTTATA ATATTTCATT TCATAAATGA AAAGTAAAAT AAATTAAAAA 240 ATAGAAAGGT TTGAGCATGA TGGCCCAGTG GTAAAGGCCA GTGGCTCCAA CGCAAGTCCT 300 GACAAATGGT AACGGGCCTG TTCTTCAGGC TTGAGGGAAG TTTATTGATT GAGGCTAAAA 360 GCAACCCAAA GGCTCCACTT GCCTAGTGTG AAGCCCTGGA TGTGCTCTCC CACACTGCAT 420 GTCCACCTGT GGTGTCAGCA CCTGGGAAGC TGAGGATGAT GGGGAGTCCA AGGTCATTAG 480 CTACATAGTA TAGGCTAGCT GGGGTACATG GGTCACAAAA AAGAAAAAAA AATAAGCACA 540 TTGTAATCCC AGCACTTGAC AGACCAATGG GGGGGGGATT GCTGTGAGTT TAAGACAGCC 600 TGGCCTACAA AGAAAAACCC TACCCAAACC CAAGAAAAAT GAAACCAGTA ATATAAATAG 660 CTATTTTCAT TTTAAATGCT CTAAAGACAC AGCGTTAACA CAAAAGCTCT CGTCTGTGGT 720 TCCTATTCCC TCCTTCTCCC CCAGGTCTTC TTTAATGTAT ACTTTTTGTT TGCTTATTTG 780 CTTGTTTTGG ATTTTGGCTT TTAAAGACAG GGTCTCACTA TGTAGCTCCA ACTATTTGGG 840 AACTCACTAT GTAGACCAGG CTAGCCAGGG ACTTATAGAG ATCTACCTAC CACTGCCTCC 900 CAAGTGCTGA GACTAAAGGC ATGTGACACT TTGCTTGGTT ATTACAAACA TTTTAAAAGA 960 ACATTTTGAA CATTAATAGA TGTATGTATA TATATCACTC TATGTAGTAT ATATGTTAGA 1020 CATTTTTCAC TTGAGATACA TATTTACTCT CAAAATAAGT TTTTTGTTTT TTTTTCTTCT 1080 TTTTAAATTT ATTTTATTTT TTTTTTATTT ATTTTATTAT TATATGTAAG TACACTGTAG 1140 CTGTCTTCAG ACANACCAGA AGAGGGAGTC AGATCTTGTT ACGGATGGTT GTGAGCACCA 1200 TGTGGTTGCT GGGATTCGAA CTCTGGACCT TCCGAAGAGC AGTCGGGTGC TCTTACCCAC 1260 TGAGCCATCT CACCAGCCCC TTAAATTTAT TTTTATCTTA TGTCCATTGG TGTTTTGCCT 1320 GCATGTATGT GTAAAAGTGT CAGAAACTGA AGTTACAGAC TGTTGTGAGC TACCATTGTT 1380 GTGGGTGCTG GGACTTGAAC CTGGGTCCTC TGGAAGAGCA GTCATTATTC TTAACCACTG 1440 AGCCATCTCT CTAGCCCTCG TTTTTTAGTT TTTTTTTTTG TTTTGTTTTG TTTTTTGTTT 1500 TTTTAAGATT TTCTTATTTA TTATATGTAA GTACACTGTA GCTGTCTTCA GACACTCCAG 1560 AAGAGGGCGC CAGATCTCGT TATGGATGGT TGTGAGCACC ATGTGGTTGC TGGGAATTGA 1620 ACTCCAGACC TTTGGAAGAG CAGTCAGTGC TCTTAACTGC TGAGCCATCT CTCCAGCCCC 1680 GTTTTTTAGG TTTTTGAAGA CAGGGTTTCC TGTGTAGCTC TAGCTGTCCA GGAACTAGCT 1740 CTGTAGACCA GGTTGGCCTC AAATTTAGAG ATTTGCCTGT CTCTCTGCCT CTCGAGAGCT 1800 GGGATTAAAA GTGTGCAGCC CAACAATCTA CTCAAAGTAG GTTTTGAAAA AGCTTTCCAT 1860 ATTAGGAGTT AACTAGCTTC ATTTCAGAAA TACTGCATGG AATTCAAATG TGGGACCATT 1920 CATAGCTACT TTGGTTTTCC TTCAGTGACA GGCATTCGGC ATGCCTATTA GGGAAGTCAA 1980 ATGGCCTGGA GAAGTCATCC TGGGTGAGAG GGCTAATGCA TTTTCAGCTT GACAGACACT 2040 GTCAACCTAT GCAGACAGTC TGCTCCAGCT CAGATGTCAA TTGCATGCAG ACCTGCAGTC 2100 AGACGCTAAG CTCCCTACCT ACTCTCCATC AGCTTAGATG TAAGGGGTGC TGGAACAAAG 2160 GCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTT TCTTAGAATT AGTATTCTAT 2220 TTTATTTTAT GTAAATTGGT ACTTCACTTA CATGTATGTC CGTGTGAGGA TGTTGTATCC 2280 TCTGGTACTG GAGTTATAGA CAGCTGTAAG TCGCCATACA GGTGCTGGGA ATTGAACCCT 2340 GATCCTCTGG AAGAATAGTC AGTGCTCTTA ACCCCTGAGC CATCTCTCCA ACCTCTTGCA 2400 TATTGAGGAC AGGGAGGAAT CACAAGCCAT GTAGGGTGCC TGGGCTCTGA GGTCAACAGG 2460 ACCATAGCCT CCTTTCTTTA TGTGCCTTTC TTGGGGTCTC CCTATAGGAG TCGTCTTCGT 2520 TGCCTCTTTA CTGTCTCATT GATCTGGGCT AAACTTATGC AGTTGGAAGG AAAGATCAAG 2580 CTGGTCATGT TTAAAACATG AAACAGCCTC ATCAGTTCCC TTCCTGTTCC CGTCTCCCCC 2640 CCCCCTCCCG CCCCCATTTT GAGAGGACAG GAAGGTAAAA TACCAAAGTG TCCTATTTTC 2700 CTCCAAATAT CAGGCTCAAA GGACTGAAGA GCTGACTTCA GATCCCAAAG CCACTGTGTT 2760 AGGAGGCACC TGCTTTTTAG GTCCTAAGCC TTCCTGAGCC TTGCTATTGG GTATTCTTTA 2820 CCAAGACCCT CAAGGATCTA GGCAAGAACT GGGCAGGATC TGTATGTAGC CCATAGTTAG 2880 ACCTAGGGCA GCTGAGACGC CAAAAGGGAG AGTTTCCTGA GGACAAAAGT GTTCAAACAC 2940 AACTGGGTGC TGGTTGTTGG GCTACTCGTG GAGGTGTGGT GTGTGTAAAG GAGGCTGTTG 3000 AATTCCCAGA AGGCTGGTTC CACAGTGTAG AGTCTACACT GGGGACTTCC CGAGACGCTG 3060 AGCCTCAGAT CTAGCTTCTC AGTCCAGGCC AGCTGATGTG GGGCTGAGGA ACAAGGATGG 3120 ATGCCATCTA TGGCCCTGCC TTGCAGGTGC AAAGGGCCTT TGGCACCATC TACAGATTGA 3180 GGGCAAGACA GGGCTGGTTC TTCCTCCTTG CTCTCGCTGC TATCTGCCTC GCCTGTAGGC 3240 TCTCTGGGCT CCTTTTTGGA CTGACACGTC TGAAGGAGCT TGGAAACTGT GAGGTCCAGG 3300 CCCCATAGAG AATCATGAAG GAACAGGAAT TCAACTGGAG CTCCGCAGCT GGTTAGGCCT 3360 GCGGTCACCT GGAAACAAAG AGGCCATTTA TTTTTTCCTT TGGTCTTGGA CAAGGAAGAG 3420 AAGGGGCTTT CTATAAATAG AAAGACAGCA AAAAAGAAAA TAATAATAAT AATAATAATA 3480 ATAATAATAA TAATAAAAAC AATAACAAAG CCAGCTCTTC CAGACAGTGC TCATGTCTTT 3540 AAAGGTCTTT AAAGGTCTGG AGTTCCCAGC AATTAAGTAA AGGACCAAGA CCTCAGGGGT 3600 CCCCTATCCT CAGCCCGTGG GGAGGTGGGA ACCATACATC GATCCCTCGG TTTATATATA 3660 GCCTCATCGC TGTGGGGCTC CGAGGTTGCC CCCAAAATCT TGCTCACCTG GAGGACCCCT 3720 GGGTGTCCTC GCCCAGAGGG CGCTGCAGCC TCGCACGTAG AGAACTAACA TCGCCCTTCT 3780 CCAGGGCAGT GCCTCCGGAC TCCGGACCAG GACATAGTAG CGAGTGCACC TGGGTCTCCG 3840 TCAGCTACGC ATCAAGGAAG GTGCGACGCG GGAATTACAG ATTGCCGGCA CTCACCAGTG 3900 CTCAGGGGAG GAAGGTGGGA CTCAGACCTG CAAGAGCTGA AGAGTGGGGT GGGCTTCGAT 3960 CCTAGGAGGC GTGGAAGGGG GTGTGGCTGG ATCCCTGGGG GGTGGGGCGC TCCAAGGGGC 4020 GGGGCAACCC AGGGGGCGGG GCCCGAGGGG TGGAGATTGG GACTACCCAG GCCCGCGGAG 4080 CTGGGGGTGG GCGGCTAGTT TTGCAACTGC AGAGCCTTTG GGTTTATTAT AAGGCGGAGC 4140 TCCGCGGGAG AGGTGCGGGC CAGAGGAGAC AGAGCCTAAC GCAGAGGAAG GGACCTGGCA 4200 GTCGGGAGCT GACTCCAGCC GGCGAAACCT ACAGCCCTCG CTCGAGAGAG CAGCGAGCTG 4260 GGCAGGAGCC TGGGACAGCA AAGCGCAGAG CAATCAGCAG AGCCGGCCCG GAGCTCCGTG 4320 CAACCGGCAA CTCGGATCCA CGAAGCCCAC CGAGCTCCCG CCGCCGGTCT GGGCAGC 4377 ATG AGA CGC GCG GCG CTC TGG CTC TGG CTC TGC GCG CTG GCG CTG CGC 4425 Met Arg Arg Ala Ala Leu Trp Leu Trp Leu Cys Ala Leu Ala Leu Arg 1 5 10 15 CTG CAG CCT GCC CTC CCG GTGAGTGTGG CCCGGGGCAG GGCTGGGAGG 4473 Leu Gln Pro Ala Leu Pro 20 CGGCGGGAAG CCGGGACTCG CCACTCGCCG ATGCCATGCA GGCGGCAGCA CGTGGAGGGG 4533 GAGGGGAGCG GGGACTTCTT CCCGCGCTGC CTGGCGGATC CTGGGATGGT GAGCCCTTTA 4593 ATGAGGACTC CTGTCCCAAT TCCTCTACGG TCCGTGGATG CCAGGAGGCT ATCCCAGCTC 4653 GTGGTCCGGG CGTCCTGCAG AGTGGAACCT CCATTGGTTC CCCGCTCCCA ATTAAGTAAA 4713 ACGACTCCAC AGGGGTCTGA GTCGCCGGCC TTAGGCGCTC CGCCGGCCTT AGGCGCCGCT 4773 TGGAGTTGCT CTCTCCCGTT GCTGTCTTGC TGGCCATCTC AGCGGCCTGG CCTCCGCCAG 4833 TGTCCCGGAG GATGCAGTGG CCATGGCCAA ACGCCTTTTC CATAGACCCT AATTCAAACC 4893 AGACTGCAGG CTGCACCCCC AGCGCCGCGG AGTCCGGGCG CTCGGCCCTT TGCACCGGGG 4953 CAAGTTTGGG CACAGCAGAG CCGGCGCGGG AACAGGGGGA AGCTGACGTT CGGGGTGGCG 5013 GGAGGGACGG GATTAAGGCT GTTTGTGGGA CACAAGAGGG TGGCTCAGGG ACTTCGGTTT 5073 TTCTCTGGCT GCCCCAGGTG AGCCGGGCCG AGCTGGCAGC GGGAGGTTCC GGGAAGTTGG 5133 CTTCAGAACG CTGAAGACCC TAAGAACCCA ACTTTGGGGT CGCTGAAGTT GTGCTGCCCC 5193 CGGAGGGCCT CCTCCGCATG GCCCGCGCGG GGGACCCTCC CCGCGAGTGG ACCCCGGTAC 5253 GGCTCTTCCC CTCCCCCGAC TCGGCTTTGT GCTGAAGCCG CGCGTAGGGA AGGCGGGTCC 5313 CTTGGCCCGC CCAGTAGGGC CGCGGGGAAA GAGGGACGAA CGTGGAGCTG GCGACTGGTG 5373 GGGGAAGCTT CTGGGTAGGA TGCAGCCATC CACCTTTGGT GGGGTCGGTC TCTCTAATCA 5433 GCGGCTTGGC GACAAAGAGC TTGGTCGAGG GTACCCCAGA AAGTGCTCTC CCGCCCCAAG 5493 CCGCCGTCGC TAGCCCGCCT TCCCAACGGG CGCTTTGTTC TCGGCCCCTG TAACCCTTCC 5553 CTGGGAACCG CCCCGCAGCG CTGGTCCTTG ACGTGGGCCG GGTCCTGGGT CGCCGCCAGT 5613 GTCAGCGCTG CCCTCCGGTG TCCACGCCCC TAGCCCCCGC ACCCGCTGTG AAGTCCCGGG 5673 TGTCCTTTCC ACTGGCGCTT TGCCCAACCC CTGGAAGGCA GAGGCGAGGT GCGGAGCCTC 5733 AGGCTTTATC CTCCCGGAAG TGGCAGTCTC CCACCGCCAC ATCTGGTCTG CTTAACTTCG 5793 ATAGTCCTGG CAAAGGCAGA CACGTGCACA GGGAAGGAGA GTTGAGCGCT GGTAGATACC 5853 AAGGTCGTGT ACAAATAAAG TGGCACACGA CACGTCCCCA GTCACTGTTA ATGCATTGCC 5913 TTCGCTCCTT CCCAGGTGGC TGGTGCTCTC CATCACTCTG GAGCCCAAGA GAGGGCCTCC 5973 ATAATTGTAT TGCCCATGAG TTGGGGTTGT GTGGGGGCGC CAAATCAGGG TTCTCTGGGA 6033 GGGCTATGAA TTCCGAACTG AGTCTCCTGT GCACTCCTGG CTTTAAGGTT CAAGAAATTG 6093 TTTGAGGGTT GTGGTTTTTG TGGGACTCAG ATTATGCCTG GAATCATAGT TACCACTGTG 6153 GAGAAGAAAG TGGAGCTACT TAGCATGCCT CCCCGGCCCG CCTGGCATTA CCTCCGGCTC 6213 TGTTCTCTAG GCCCAACGTG AGGCCTCACT GGGGCAGTAC AGATGCAGTA CTGAATTTCT 6273 TTCCAGCCAG GATCTGGAGA GGTGGTGTTC TCTTCCCTGG TGTCTTTAGA GAGGCAGATA 6333 TTCCTGTGAC CTAAGCCCCT CAAGCACCCA TTAATAATGC TGAGTAGACA ACTAGAGGTG 6393 GCGTTTTCCG GAACTTCCTG TGTGCTGGCC TGGGAGGTTG AACCCTCTAG GAAACAGGTC 6453 TAGGAAGTAG AATTATCTCA ATGGAAGGCT TCCTGGAGGA AGAAGATGAG CTGAGCCCCC 6513 AGGTCACTGT CTGAGCTTTA GGATCAGACT CCCACTTGGA GGCAAGAGTG TTCGTTTTAC 6573 TTTTTTTTTT TAAGTTTAGT TTATTTTCTC TCTAACAGAA AACAAACAAA CAAACAAAAA 6633 AAAACCCCAC ATTGTTTAAA AGTGGGTGCA TAAGAGTGAG GACATATTCA GAGCTTCCCC 6693 TTTTCCTGAA AAATGAAGGC AGCTGGGATT TACTTAAAAT GAGAGCACAT ATCACAATTG 6753 CCAGAGAGCT GGTCCCTTTC TCAGGGCTCC CTAAGCTCCT GTGGGAAGCA GGTCAGACAG 6813 CCCTGGGGAC CAGAGAGATA GGGAGTGCTT TTGGGTGCCT GCCTTTGAAT GGGGAAGGGG 6873 GGGGGAGCTG CTGGGATCAG AGGCTGCTAG CAACTACTCC CCAGAGACTG AAGCAGGTTT 6933 GTCCCTCAGT GTCCTGTGGT CTTCTGTTTC TCCTATATAG AATAGGAGAA ATGGTTATTT 6993 GCTCTGGAAT AGTGACTTGC TATTTGTTCC CTTTCTTTCC TCTCCCTTAC TGTAATCATT 7053 TGGACTAGTA GAGACACTTT CCCCAGGTCT GGCAGAATGG GAGGGAGTGG GGGAGGCCTG 7113 TGCTTGCATG ATGTCACTGC TGGCTTCAGC TCTCCAGGGA GGGTGGAGTT GGTTGTAACC 7173 TACCTGTGGC TCTTGATGGG CCACAATAAA ACCTCATTAA CACACATTGG TAGGGAGAAG 7233 GGACTGGAAA GAATGATGGG AAAGATTGAT GTTTTTCCTT TTTTTTTTTT TTTTTTTTTG 7293 GCAGTACTTT CTAGATCTCC CCTCCCCCTT GCTGCAGCAA AATTTTGGAT TCCTGAAGTC 7353 CTTTGAGAAT GTATAATGGT AGCCAGACTT TTTTTTTTTC AGTCAGCTCA AAATTGCCTC 7413 CTTATAAAGT ATCCTTGGTT GTTTTTTGTT GTTGTTGTTG TTGTTGTTTT GTTTTGTTTT 7473 AAGACAAGGT TTCTCTGTAT AGTCCTGGCT GCCCTGGAAC TCAATATGTA GACCAGGCTG 7533 GCCTCAAACT CAAAGAAATC CACCTACTTC TAACTTTCAG TGCTGGGCCT AAAGGTGTAG 7593 GCCACCAAAA GTGCTCAACT TTTACAAAGC AGTCTTACTT TGAGCAGGAT TCTGAAACCC 7653 TTATTTCCTT TCTGTTATCT TCAACAATAC ACTGCTAGGT GTATTTAGTC CCTCATGATG 7713 CTGGGCCTCC TCAAGTGGCG CCAGGTCAAG CAGTCTCCTG GTTTTTGGTG GCTCTGAAGA 7773 AGACTGTGTC CCAGTGACTG GCAGTTTGAA TTCGGAGCTT CTCTTTTCCT TCTCAGTCTT 7833 TGGCAGGCAG AGTGACACTG GTGTGCCCAA GCCTGGAGCT TCTCTGTTTA ATTCTAGTTT 7893 ATTTTCTTTA TCAGACTGAA AAACAAATCA GGTTGGTTAT AATTCTTATA AACACGAAGG 7953 TCTCACCTTT GCGTACGTCT CCGGCTGTGT GGGTCTGATG TCCCTCGGGA ATCTCTGTTG 8013 AGGCTGCTGC AGTGTGTGTG CGTGTAGAAA GGGCAAGGTA GAATGGACAG AAGCGTGCTG 8073 CCCACCCCAC TGTCCTGTTC CTAAATGATG AAGCACTGGC CCGGTGAAGA GCCTAGAGAA 8133 CTCCCTCGGT GGGAGATGCA CACAATGCCA GGAAGCACAC AGGAGCTTGA GTTCCAGCTT 8193 GGCAGTGTCT TCTCTTTGGT GACTTTATCA GCTCCAGCTG CCCTGGACTA ACAAACAAGG 8253 CTAGCTCACT CTCAGTATTG ATAATCGAAG GTCCTTGGTT CTGTTTGAGA CTGATCCTCA 8313 CTCGGTAGCC TTGAACTCTT AGCAATTCTC CTGTCTCAAC TTTCAAAGAG CTGAAATTAC 8373 AGACTCGAGC CACCATATGC GACTGAAACC TTGTTCCTAA TCCTTGACTG TGAACGACTC 8433 TTGGGTTTGG TTCTTTCTCC ATTTCTTTAG TGTATGTTTT AGTTCGCGTC CTACATAATC 8493 TATTGCCCAT ACTTAGAAAC AACAGGTTAG AGACAGCATT GGGTCCAGCA GAGCCTCACA 8553 CTGAAGCTCA GTCCTGCCAC TGATTTACCG TGTCAGCTCA AGTGACTCAC TTCCAACTCC 8613 TCTGCTCCCC ATCTGTAGAG TAGACATCAC CATACCTGCT CTTTCTGCCC ACATTCTGTC 8673 ATTAACATGT TCATTTCATA ACGATGGTGC AAAAGTGCTT TGTAAGTAAA GTGCTGGGGA 8733 AATGTTAGCT GTCGATAATG GTTAGGGTTA ACTTTTTATT GAGTGCCTGT TGTGTGTGGG 8793 GTTGGGTGGG GTTTTTTTAG AGGCTTGGTA GTTTTCTTAC TTCTTTCCTA CTTAGCTTTT 8853 CTTCCTAAGC CTTTATGGTA TGTATCATTG CCTGATTGTT TGAGTGTGTG CACTGAGGCA 8913 CGCCTGTGCA TGTTTGAGAG TATGCTTGTG CGTGCTCTCG TGCTCACATA TGTATGGTGT 8973 GAATACACTG TAGAGTGCAG GCCGGCACAC TGGGGCTGGC TGAATCCTGT GAGCCCTGCC 9033 TGGAGTTTGC AGATCTTCCT TGGACACTCC TGCTTGTGAG CATTTTGTGT GGAGTGACTG 9093 TTTAGCTGGC TGTAGCCTAC ATTGTGCCTT TGGGTAAACC CTGAGTATTG GGAAACACCC 9153 TGGGCTGTGG CTGTGTGTGC CCGACGGTTG CTTGGGTACA GCTAAGAACT CTTCATAGAA 9213 AGTTGAGCTC ACATGCTATT AGTATTAACT GAGTGCTAAG GAACCTGTCT TGGGTGGTAC 9273 CTGCTTGCCC TCTCATGCAG TTTATCTTGA GCTTGGCGAA CACACTTACA GATTTAGTAG 9333 AGCTTTTGTC AGCCCTGGGA GGTGGGTTTC GTGGCCACAA GTGGGTAGCT TGGAATCCAA 9393 GACTCCTGGC TTCTAGGTTG CATTCTCCTG TGGTTCTTTC CAAGGGAATG CTAGGGGAAC 9453 ATTTTGGACA TTAGATTATT TCTAGTCCCA AAGCACACAG AACATACTGT TTCCTAATTG 9513 CCTTTTTTTT GTTTTCCTCT CAATCTGGTT TTGAAGTGTT GGGTTTGAAA ATTGCCCCCT 9573 GAGAGCCTGC CCTAGTGTGT GCAGAGGGAA GATAGTGGAA CAGGAAGTCT GTAGAAAGTA 9633 TCTTCCTTTC CAGGACCTTG TGCCCCGGAG CAGAGTCAGC ATGGTGTCAT ATCGCTTTTG 9693 GCTATTCCAG AAGAGATGAG GTTTTAGGTG AGAATGAACC TTTTAGAACC TTCTAGAACC 9753 TTCTGTTGAG TATGACAGGA ATGCCCTGAA TAGGGTCCGA AGTGCATGGC CACTTGTTTG 9813 TCTTTTCCAT AAGCAAGCAG CTTCAGGTAC AGACAATAAG ACTAGGTTCT TGGAGTGAGA 9873 CCCTGCACTT GGTGCCATTT CAGCTCCAGA TGGACACTGG AGGTCCCTAC ACAGCAGGCT 9933 CTGGGATGGC TGGCTTTGCT ATGTACTGTT GCCTGCTCTA CAAGAGCTTC CCAGGTTACT 9993 AGCCTTTGTC GACGCTGGGC TCGCTGGCCA GGCTTGGGCA TTGGAGAAGG GACAACTTGC 10053 CACCTGGCAT AGGCTGTGTG TTTGGAGAGT CAGGAGGTCT GGTGAAGCCC GCAAGTGGAG 10113 GCAAGTTTAG TGGGACTTGA GGAGAGCTCA GTAGGAAATC TCTGGGCTAG TGACAGGCAG 10173 GTGTGGTGGT GGTGGCGAGG TGGCGGGTCT AGATCTCCTT TTAGAGATTT GCCTAGGGAT 10233 CGTCCCTGCT GACTCTGGAA CTCAGAGGCC TCCAGAGGTG TCTCCTCTGG GAGCCTCTCA 10293 AGGGTCTCCC ATCTCCTACT GTTTATGGCT TTGTGGGCTA CCTAATTACA TAGAGAAGAT 10353 ATGTTCCTCT GCCTCCAGCC CTGGAAAGTT CTGCCCAGTG ACTCACCTGA GCCTGCAGCC 10413 ATGTGTGTAC ACAGGCGCTC TCAGGGGCTT CTGTCCTGCT GGCTTCAGCC TTTCTAGCCC 10473 CTGGTGTTCT CGGCAGTGGT AGCATCTGGG AAACCGGGTC ACCTCTTATT TGCAGCTCCC 10533 TCCCTTTCTT GGTGTCTTCC CCCTTTTTAA CTACTGGTCT GATGGCCTTA GACTCATGCT 10593 GAAATTCTCC TTTCTTTTGT CCTAGCCTTG TCTCTGACTT CTTGTGATCC TCTGGGCCTG 10653 TGAAATCCGC TCAGGGGCCT CCATTTCTAA CAGTCACACA CTGGTGGAGA GACCGAGTCC 10713 TGGGATGGTG AAGCTAACCC TGCTGGGCTT CTCAAGCTTC ATTTGGTTTC TCTTTATTCC 10773 TTCTGGAGGT ACTGCCTGCC CCAGGGGAGT CTCAGACTAG ACCACTCTGG AGTTGGAGGT 10833 GGGGCAGGTT TTCAGATCAG TGCCCTTGGC ATTCGTTGTG GGAATGGGGT GGATGGGGCC 10893 TCTGGGCAAG GTCAGGCTGG GGGTGGAGGC CAGGTGATGT TCTCCGCACC CACACCCAGG 10953 CAGCCTGGCA CCCTCCCCAA GGTCCGCTCA TCAGCAGGAA TGAAAGCAGT GCCGGGCAGG 11013 TTGGGGCAGT GGGCAGGTGG GCGTGTTTAT CGCTGTGCTC ATCAGCTGAG TCACGATGCC 11073 AGGCCCCACA AGTCCTCCCT GGAGGCTCAC CCCACCCACC TTGACCCACC AGCACCCACT 11133 AGCAGGAGGT AGGGCAGGGC AGTGAGACAA GACCAGCCTG GGGGTCTGAG AGGCAAAGGG 11193 GAGTTGTTCA TGACCTGGCT GTGCATGGGG ACTTGTGGGT GTCTCAGATA TCTCTGCTGT 11253 CCAGGAGGAA GCTGTCTTAA GTGCCAACCT GCCTAGAGCC CCTGCTGGGT GCAGGAAATG 11313 CACAAGGGAG AGTGCCCATC CATGGAATAG GCCCATGGAG CTAGACCAGT GACAGTGACA 11373 GTGAAGTCAG CCCCCACCTG TGTCTTCCGA GCCAGCTGGA GGGTTTTTAT CTCAGATTCT 11433 GCGAAACCAT AGAATCTAGT CAGGAGCCTA GACTGCAAAG CAGGCTTCGT TGATGCTTTA 11493 ACTTGCAGGC TTCCTGGGTA TGAGGGATAC TTAGAAAGGT CCCGCAGGTA GGGAGGGCAT 11553 CAGGAAGTAG AAGAGGGCCA GGCACTTCTA TCTCCTGCAT TGCCCCCTTC TCCCATCTCC 11613 AAGGATGGTA AAAAGAACCC TTCCAGTACA CTGACAGAGA GGAAAACCCT TCATCTCACC 11673 CCATTTGGAT CTGTCGTATC AGCATGTGCT GGCCCTGCTT CCATACCAGA GGTGGCTAGA 11733 GATGTTCCCT GGGAATTCAC TGGTTGGGGA CTTGAGTGTA TCAGAGGGGC ACAAAGTAAC 11793 ATTAACTCTG GTATCCTCTG CAGCAAATCG GAGATCCCCT CTCCTAGGCG AGTTCTCAGT 11853 GGATATGGAG GTCAGGTTTG GGCTTGTAGG GCCCCAGCAA GAGTCGTTGA TGTCACTCCA 11913 GCTTCTCCCG AGGAAGATGA GGGTGCTGTG TTGGGATCAC ATCTCTCCCT GAATGGCATG 11973 TTGGGGAGGG ATGGAGCCCT TGCTTCTGAC CCCTAAGCTT GGTCTTTAGG TGGCCACAGT 12033 CTCTGGGTTC TGTCCTACCT CCCTGCCCTT GTGTGCTTCA AAGGCATGCT AAAGGGACTC 12093 TCGGCCATTC CGAATGGCAC AGTGTTCCTT CTGTTCTCCC ACCCCCAGAA GGAGGCAGGC 12153 CTGGATTGTA GATTCCTAGA AGTAAGTGGC CCTGAGCATG CTGTTGATGA ACCTGGAACC 12213 AGGCAGGCTG GGCATCCTAG GACCTGTCTT TCCATAGAAG TCTGAATCAG TCTACCTTTG 12273 GGACTGAGTA AGGGGCTCCT CACATATCAG CTGGCTAGTC CATCTTGGCT GATCTAAACC 12333 ACATTAGGCT GAAGAGAAGC ATGGTGTACA GTCTGGTCCA CCCGAACCAC ATACTGGCTT 12393 TATCAGTTCT CGTATAATTT TGCAGGTAAC TTTTTAGCTC TAAGCCTGTC TCCTCATCTG 12453 TGAAATCGGG TCCCTCATAT CCTGCCTAGA AGGGCTTTTG AAAAGATTAA TGAAGTAGTA 12513 TGCCGAGTGG TTGGGGTTCT CTCCTTGACT GGAGCAAGTC TCTAGGAGTA CTAAGGATAG 12573 CCTGCTGTGT GCAGCACCCC CAGGGACTGT GCCTGAGTAG GAGGGTACAG AGTCTTCATG 12633 TGAATGGCCC TTCTGGTCTT GCCCCGAAGT TAGTGTTGAT GTCATAGAGT CTACAAACAT 12693 GCCTTTTGTC CTTCCTCAGA AGTCCAAGCC TTTCCTGGCA GACCAGACAT TCATCTCCAC 12753 TGAGCCTCTA TGTGAGACTG GCTCCTGGCC TGAGCTGTGT GGGCTGAGCT GGCGAATGGG 12813 AAAACTAGAC ACCTGGGCAC CTGGGTGGGG GCTCGGGACA GCAGTGTTTC AGTTGTAGGC 12873 ACTGTGCCCC TGCCTGGAGC TTCTGACTGA AGGTTACCCT GAGAGGAAGC AGGTTCCCTA 12933 TAGACACTAA CATAGCTGGG TCAGAGTGCA AGGTGGGTGT GCCCCTGCCC TGACCCATTC 12993 AGTGCAAAGG CTGCTCTTCT GGGAGTGAGA GCTCTGACAG GACTGTGATG GCCGAGGGGT 13053 CTCAGAGCAA ACCTGCCTGG CCTCTCCCCA CTCTGATGGA TATGTGCTCT TAAACAAGTG 13113 ACTGTCCACT TTGCCTCAAT TTCAACATCT GTAAGATAGA TAGGGCGTTA TGGTCTGAAA 13173 ATGGTTTTAA AGATTAGTTA GCTAATACAG GGAAAGTGCT CTGACAGGTA CCTGGCACCT 13233 TACTCAACAA GTGGCTGGAG TGCCTGATTT CCTAAGGTCT CGACCTGTCC CTATGCTTCA 13293 AGTGCCCCTA CAGCCTTGGT CAGGCCCTTA GGTTCTCCCA CCCACCGCTG GCCCCAGGAC 13353 CTAGACTGCT GGACCCTGAC CCCATTTTTC CTTTAAGCCA CCTCTGCGTC AACTCTAAAA 13413 GGCGGTGGAG TTGTTTATCT AGGCTGTGAG GTGTCAGAGA AAGGACCTGG GCCGCTTTGT 13473 TCCTGTGTGG GCTGGGGCCA CTCCAGGAAC TGAGAAACCC ACCCACCTTT TCAAAAACAG 13533 CCTCTTCTCA GAGTCTGGCA CCTCAGCTAG CCACCATGCT GTGGGACCAC TCCCAGCATG 13593 CTCTGCCTTT GGTTTGTTTC CCAGGGGCCT CAGTGCCTTT TAAAGATGCA CAGGCATCTT 13653 TAGTTCAAGG GGAAAGAGGA AATGAAGTGT ATTTGCTGGT GGTGGTATTC CTGTCACTTG 13713 CATTCTCACA GAGGCTAAAG AAATTTGCTC TTTGTATCTT CTAGTCTCTT CTTTATGATC 13773 TTTTCCCATC TGTTGTATCC CAACTGCAGG GCCCCAGTTC TAGAATTAGC CCCTCCCCCA 13833 TAGGAAGCCG ACTTATGCTA TAATGTGAAT GACAAGTATC CTTTAGCCCT TCCCACAGGC 13893 ATTTTAATTT TCAAAAGGGC ATTGCACAAC CGCAGAGACA CTAAGAAGAG AGGTTTGGTG 13953 ATCAGAGTTA CAGCCCCAGC CTCCCAGCTG GTGGCCCGGC TGGTGCAGGT GTGTCGAAAG 14013 CAGTAGTTTC TGCTTCAGTG AAACTTGAGG ATCCTTTATT TAGCCAGTTC AGGGGCGGAA 14073 TGGCCATGCG AAGTCTATGT GTCACAGGTG TCAGGCCCCC ATATCCTGCT GAGTCTAGAA 14133 TCAGCTACGT AGCAGTTTTG GGGGTATTGC CAGACTGGGA GTTTACATCC CAGAAGCGAG 14193 AATGGTGGGG TTCCTATACT GCTCCAGACA GGATCTTTCC CCCAAGTTTG TCAGCCACCT 14253 CTCTTCAAGT CCCTTGGCTC TGACCAGCAA GACGTATCCA AAAGAAACTG AGGAGGCCCT 14313 TCACTTCTTT TTAGGATAGT GTGGGGCCAG CATGGTGGGG GTTGGGAATG GCTTTCTGTC 14373 TCTTCCATCA TCACAGGCTA CTTCCCAGAG ACACTTTGAT TCTGGGCATC TCCAGCAGTC 14433 ACCTGGCCCA CAATGCTTTG CTGCCCTTTG CTTCAGCCAC TGTATCTGGT TGTCCCTTGA 14493 AGGTGAGCCA GAGCTCCTAG GCAGAGAGCA TGTGCTATAC AAAGCCGTAG GCTGGGCCCT 14553 GGGAACCTTC TTGCTGTCAT CCTCCTGTCA AACCCCTATG GTATGGTAGC CCACATAAGG 14613 CTTGTGCAAA AAACAGGCCA AAACATAAGT TATCTTTTCA CTCTATCGGG TCTTCTCATT 14673 TTCCCATGGT ACGTTCGGCT GGCCAGGCCC AAAAGATTTG AAGAGAGGTG GCTGGCAAGT 14733 CTAGGGGAAT AGGTCTATCT GGTTCCCTCC AGGAGCAGTG CCTAGTGAGA GGCTGGGCTG 14793 GGCAGGGCAG GGCCCCTTGC TCCACATTGC CTGAAGTCCC GCCCTGCCCG TCCTGGCTGG 14853 GATCTGGCAG GTCTTCCAGC TCCACACCCG GCTCTCAGCT GAGCCTGCTC AGAGACTAGT 14913 CCTGGCATGT GGGTTGCAGG GCTGGTTCCA GCTCCACCAG GAGGTATGGG CGTCTGGGTA 14973 CTCATGGGAC ATTGACCTGT AGTGGGTATG GAGAGTGGAG GAATGGTACA GGCAGGTGTG 15033 CTGGTGCTGA CGGACTTGAC TCCGGCATTG ACCTTGGCTT GCAGTCTGGT GTTAAACTAA 15093 CAGGGAATGC TGACAAAAAA GACAGTTATT AAAACCAAGA CAGGATACTG CTTTCCCACT 15153 CAGCCCATTC CCAAGAATCC CCAAGACGTA CAGGAAATGT GCAACAGCAG TGGGAATTGC 15213 TGAGTTGGGG GATGTGGGTG AGCTGTGTGC TCCCAGGGAA TTTTGGGAAA TTCCCCTCCG 15273 TTGAAATGCT GTCAGGGTCT GAGCCTTGGA GGTGTTTTTG GGGTGCTGTG CTCCCCAGCT 15333 AAGCAGCTAA CAGTCCTCTT TACCTGCCTT GTCCTCACCT TGCCCCACCC TGGGTTGGGC 15393 CTCTCGTTCA CTCCCTGCTG GGTCACCAGT ACTTCAGTGC AGGTCTCAGC TTGATTCTTG 15453 GTGGAGAGAG AGAAAGTTGA TAAATCAGGG TGCCTGTCAG CCGGAAATTT GGGTGTGTCC 15513 TGAAGGCACC AATGGGGGCC CTCCCTTCTG GAGGTGGCTT TAGGAAGGGG TTTCTGGGTC 15573 TTGAGGCCTC CTTACAGTTT CTTAGCTCCA TGGGAGAGAA GTGAGGAGTT GGGTATCGTC 15633 ACCCCAGCAT GAATCTCTGG TCACCTCTCA GCATGCACTG TCCAGCCTGA TCTTTGAGTG 15693 CCATAAAAGA ACAGAATTAT CCTCTCAGAG CACTTCATTT CCCGCCAGCA CAGTGGGTAC 15753 AGAGACAAGC TGCCCAGACT CCCAGCGAGG GACTAGTTGA GCCCCAGCAT GGGACTAGTT 15813 GAGCTAGACC TGATACAGTC CCAGAGAGCC TCGTTGAGGA AGCTTTGGGA AAATTCACCC 15873 AGCATTTCAG CCAGGACTGG AGGAAAAGGT GATTATGGGA AAGAGAGCAG TCAAGACCCC 15933 AGGCTGTAGG ACACAGGATA CAAACTGAGA GCTACCGGAT AGGAGTAGGT TTTAGTCACA 15993 ATCTCTCCTG TCCGCCCTAC CCTCCAGGAG ACATTGCACC TTGTAGAACA GCTGCCCCGG 16053 AGTCCACCTT TGGGCCCCCC TGGGTAGCTC AGTAGTGTCA GCATCCTCTC ATTGACATCA 16113 GTCAGGTTAC ACAGTGGGGC AGCTAATGTG AAGGCGCTAG GCTGGGAAGC CAGCTACTTG 16173 GGAAAACTAG GTTGTTCCTG GTAGGCCCTA GCAGGAAGGC AGTTCCTCCT TTTCTTGGTG 16233 GCTTTAGGGG TCTTTGGAAG CTTTGAATGT TCCCTCAGCT CGTTGGTGAA GCAGGCCCTC 16293 CTGGTACTGT GGTGTTTGTC TTCGAAGAGT GAAGGCATTG GAAGTAAAGA CTGATGGGGC 16353 GCCTTCCCAG GATGCTTTGC TTCTTGCGCT GGCTTACAGA GCTCTCTTGC TACCTAGTGC 16413 CTTGACTTTG AACACCAGAT TCAGTCAGGG AACAGGAGTA GAGGTCTTGC CTTGCTGAGC 16473 CCCTGCGCAC TGCAGGAAAA GACTCCTCTG AGTGGAGCCT TTCCTCCTCA GGTGACTGCT 16533 TTCAAAGTAC AGCAGCCTCT GAGGGGGAAG TGTCATTTGA CATTGTGGTA GTTCTTGGGG 16593 TCCCTGGATA CAGATGTCAT GCCCAGATCA TAGGTCTGTT TGTACAGAGG GAGGCGAGTT 16653 CTGTAGCTCA GAGTCCTCAG TACCCCAGAG TTGTGGCTCT AGGGGTGAGA GGAGAAGACT 16713 ACAGCCCTTC AATCACAGGT CTGACCTGTG GGTAGGGGTA GATCTCTTGC ATACTATGAA 16773 CCTGTTTGAA ACCCCTGGGT ATTTGCTGTG GAATAGAGTC TTGGTTGGGT AAGAATGGTG 16833 GATGTTTATC TTGGTGTGAC TCTCGGGTGG GGGTGGGGGA TATGTCCCTG TCTTTCCCAA 16893 TGTAGTATGC TGAGTGGACA GAGACCGTGT GACTGAAGCC TGGGCTCCTG GAACAGGTGT 16953 GTGTTGGTGG GGGGTGGGGC GCAACTATCT GGGATCCAGA CTGCTTGGGA ATGGCTGTGA 17013 CCCAGCTCCT TTGATAACAG CAGCTCTTTG TCACTGGATG TTGTGACTAA TGGGACTTGT 17073 TGATTCAGTT ACTCGGCTCC CACCCACAGA CGCCGGGGCT TCTGTTGTGG CACCAGGCAG 17133 CTGCAGACGG CCCACAAGTT TGCCTCGCTT TCCCACTCCA CGAAGGTAAG TTCCCAGCAC 17193 TGCCCAAATT AGAGACTTGT GAGTGGTCCC CTCATACCCC ACTCCCTGAG GCTTCTCCTG 17253 GAAGGCCTGG AATGGGGCAC TGGGTGTGTA CGTGCTGTGG TTTCTGTTAG GGTCAAGACC 17313 AGGCTGTTTC TTACCTGGCT CGTACCTCCA AGTTTCCAGG TGATGAGTCC TGATTTTTGA 17373 AGTGAAGGAA TCCATTTAAT ATCAAAATTC TGTGACCTTA AATTTTTTTC TTTTATTATG 17433 TGTCATTTCA TATGTACGCA TATTTTTTTG TCTGTGTGTG GACATGCTTG TGGCGATCAG 17493 AGGACACTTC AGAAAGTCAG TTCTCTCCTG CCGTGTGGGT CCTGGGGAAT CAAATCCAAG 17553 TTGTCAGGCT TTATCCTGAA AATAAAAAGT AGACAGCCCT TGGGATCCAA AGCTTCTTAG 17613 GGCTGTGTGT CTTAGACACC ACCAGTGTTG CACAGCTGGT AACATGACAG TGTCCTGGAG 17673 TGCTGATTGG AAGCCACAGG CCTCTGTGCA GGGCGGTAGA CTTCCAGGGT ACGGGGCAGG 17733 TGGGCGTTCT CTACAAAAAC CTTGTAATCG CGGACGTCTT GGAGATGCCC CCTAGGTATC 17793 ATGATTTTGG TGTGTGACAC AGCTGAACTG TCTTCATACT CAGGATATCA TGAAGTGCTG 17853 GGGTGCAGAC CACTCTCAGC CTCAGGCAGC CAGGACCCGG GGCTCCATCA GATTGCGGTG 17913 ACTACCACAG AGGGTGGCCT TCCTTCCGGT CAGTGTGGGT GTGGGAGCTG GCAGGAAGTG 17973 GCTCCAGGCT TCCTTTAAGC ATCCTCTGCC CACAGCCCCA AACATGTTCT TTGGCAATGG 18033 CTTGCAACTA GAGGTGAACT CTCTCCTGTA CTATGTCCTG ACCCACGCTG CTGCATCTAT 18093 TATACCTTTC ACACGCGTGA TGGGTACCCA GCGGGGCTGC TAGGCAGGGT TAAGCACTCA 18153 TCTTGTTTCC TGGTGCTGAA GCTGTGGTAA AGAAACTGAG GCCATTTTCC CTTGAGAGAG 18213 ATGGTCTCAG CCAGGTCTTT CTCGGCCTGG GGAGCCCGGA AGAAAGGATG TACTACAGTG 18273 AGTGGACACT TGTTGGCTGA TGGCCTTGGT AGGTCCTTCA CCCTGGGAAG TGCTGTTTCT 18333 TATCTGTTAG AGATGCTGAC CTCAGCAGGA CTGGAGGAAC TGCATGGGAG GTGTAGGAAT 18393 GAAAGTGAGT GGGGAAAATT ATCTCCAGCC CTAGGGAAGT CTGAGGCCTG TGTCCCCTTT 18453 GTCCTGGACT GGGCCCCTGC CTTGGGTGTC TGTCCAGGGT CTTTGCTCTA CAGCCCCAGC 18513 GGATGCCCAA AGTAGACGAG TCAACTGGTC CTTTCTTTCA CCCTGTGTCC ACTTCTCATG 18573 TATCTACCTT CATAATCCTT CTAGGTAAAA CAAGCCTCTA ACTTTGGGTT TTCAAATCAG 18633 CCAGCTTCCA GGCTCGATAG TACGAACCAT GAAAATCTTT CTTACCATGA GGTTGTTTTC 18693 TAGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTAC GTACACATAT 18753 GTACCTCTAT CAGTGTGCTG TGCGTGTACC ACAGCAGACT CGTGAGGAGG TCAGGCAAAC 18813 TTTATAAAAA TCTTTTTTTT TTGCTTCACT TGAGTCCCAG GGTCACACAG TGGCAAGTGC 18873 TGAGCTCTGT TCTCTGTTCT TGATTTGTTT TGTGAGCAGC TGATGTTCTT AAGGCTTGCG 18933 GAGGGGAAAG GTAGGGCTGG CTTGCTTCTT CCCCGAGTGG CGGTCAATCC CTAGACATCT 18993 CTAAGCCGTG GCCACACGTC CTGGAAGGAC CCAGGTCAGA AGTGATACTG AGATGGCCCT 19053 GTGAGCCCTC TCGAACACAC AGGGTTGTAA ATAGTACCTG ATTGTTACAT TGGAGACTCG 19113 TCAGCTGGGT GGAGTCCTGG TTCAGAGGGA GTTATTCCTC CCCCCACATT TCTTCTCTTC 19173 TGGGGCTGAA GTCTCTTCCT TCCTTACCTG TGATGCTGTC ATGATAGGTC CCAGCTGAGA 19233 GTGGAGGCGG GGCAGTCAGG GAGCTGCTTC TCTTTGCTTA GCAGGGGTTG GAGACTTGGG 19293 GTGTAGGGGT TGGCTCCCCC TTTCCCTGCC CTGGACCTGG TTTCTGGTTT CAGCAGAGAT 19353 TCGTTCTAGA AACTTGTTGC GTAAACAAGA TCACAAAGCG ATAAGCTTGA GCAAAACCCA 19413 GGGGAACAAA TTGCTTCCCT GTGAAGACCC AATCTTAGCT CTTAGAGAAG CCCTCCCTTT 19473 TGGAAATTGC TGACTTTCAG GGCTTCTCTG TGGAGGAAAG AGGCTAGCCG CCGTATGTTT 19533 GCCTGGATTC CAATAAATCT TTGCGGCCTT GGCTACCCCT TGTTGAACAA GGTCTGCACT 19593 CCTAATGCGT GCCTCAGGTG GTCTGAGACC TCTACCCCAT CTCCAGCTTT TCCTTCCTAT 19653 GGAGGGAGTC AGTGGGTTAG GAGAGAATGG AGTTGAGTCC TGGAATGAGG AGGAAGCTAT 19713 GAACTCGGGG CCTGTTCCTG TCTGGTGGGT GCTCTTCTCC GCCGCTGAAG GAGGCAGCCG 19773 CAGGGAAGAC TACCACAGGA ATCCGAGTAC CACCTGGAGC AGTGTATACA GGATGTGGGC 19833 TGATGTGTGG TAAGGGCATG ATGGGCTGAT GTGTGGTAAG GGCATGGGAT CTGATTGCTC 19893 TGTGGATGGG CCACAGGGAA ATTTTTGAGT GTCTACTGCA GTAGTTCTCA ACCTGTGGGT 19953 TGTGCGCCCC TTGGTGGGAG TTACATATTA GATATTTACA TTATGATTCA TAACTGTAGC 20013 AAAATTACAA TTGTGAAAGA ACCAAGAAAT CACCGCAGCA TGAGAACCTG TATTAAAGGG 20073 TCACGGTGTT AGGAGGGTTG AGAGCCACTC ATCCTCTGGG TCTAGGCCAT GGCGGGCTGT 20133 AACTGCTCTC TGGAGTTAAG CCACAGTGAA CCAGCTGTCC TTGCAGATGG ACTTGTGGAG 20193 GCTCCAAACC TTTGTCCCAG GGGAGAAGAG CTTGCTTTTG CTTTGTACTT TTAAAGGAAG 20253 TTCAGTGGTC TTCGGGCCTT GTGGCTGCTG TGTGTGGAAG TGCCCCTGTA CAATAAGCTG 20313 TATAGATCGT GTACAACTGC AGTTTTCCTC CGTGGGTCCA CCAACCACTC CTGACTCCAC 20373 GGATGAGTGA GGCCAGTAGG GCTGTGTGTG GGTCCCTAGG CCAAGCATCC TGGACCACGA 20433 TGAGCCTCAG CTAGACCACT CTGGATCTTT AGCAGAGGCT CCTAGAGAGC TGGCTGGCTT 20493 CCTCCTGCCT TCTTTTCTCT TAAAACTTCG TCTCAATCGG AAGCTCCTCT GTGCACGTGA 20553 CCTCCAGGCC TGGGGGTCGC CACAAATCCC CTCATCACAA GACGAGCAGC TCGCATGAGG 20613 GACACGACAC TTGTTACCTA CCAGGCTGTG GGGTTTTTGT TGGTTGGTTG TTTTGTTTTG 20673 TTTTGTTTTT TTACTTGTAC AGAAGTGTTG TGACATCAGA TGTCAGCTGT TAGTGCTGGC 20733 ACCATTTTAC AGGTAGGGAA CTGAGGCTGT AAGATGTGTA GTGACATCGC TAAGGCCACT 20793 CAGTTGGTGA GGCCTTACCA AGGTCAGGTC TTTGGAGCCT TTTGCTGAAC CATGTACTTC 20853 TATCTCTGTT TTGTTGAAAC AAAGTCTATA TGGCTCTGGC TAGCCTATAA CCCCATATGT 20913 AGACGAGGCT GACCTCGAAT ACACTGCAGT CTTTTATGTC TGCCTTCTGG GTGGCAGGAT 20973 TGAAGGCATG TGATTCCTCC TAACTGTACA CTTTAAAAAA AAAATCATTC TTTGTTCTGG 21033 TCTGTGCCAG GGCCTTGTAA GATGTTCTGT GCTGAGCTGG GCTATTTGGG TTAGTCTCAT 21093 TGCTGAGCAG GGCCCCTGTA TCTTCCTTCT CTGTCACTTG CTTACCTGGG TCTTCCTCCT 21153 GCACTAGCTA TCCTAGAACC AGTACTGAGA GCAACTATGG GCCCAACTCT GCCCCTTGCC 21213 CAGCCTGCTT AGCTGGGGGC GGTGTTCCAC TTCCCTGCCC AAGTCCTGTG GGACTGTGTT 21273 TGTACTCCAC CACCTTCAGT TCCTTGGAGC TGGAGCAGGC CAGGCGGCTG CATTCCTGCA 21333 GCTGCTGTTG CCAGGGAGAG CCCATCCCAT TCACTTCAGT CTCCTTAATG TAGAAGCCTT 21393 GTCGAATTAG CTTCCACTGT CCCCAACCCA AGAGTACCCT GTCCTTTCTT CACTAAGAAG 21453 GCCAGGATAC AGTCCTTCCT GTGGCTGATA AGACAGGCCT TGGGACAAGG CCTGGGACCA 21513 CACTGTGTGG GCAAAGCTGC TTCAGCACCG ATGGCTCCTC CATGCCAAGC TTGGCTCTGC 21573 TTCTCACAGT TGAGACTTCT GTGCGCACAC CCACTGTCTA GCTCAGCTGG ACACTGATTT 21633 TCTTTAAATG TATAGATTTT GGGGTGGGGT GTGCTGAAAG CTCCCACTGA TGCCCCAAGC 21693 CTGAGTCTCA GAGTATGATC AATTGATGGC TTTCATGGGT ATCACAGCTT CTGTTCCCAG 21753 GTCAGACTCC CTGACCAGTC AGAGCATCCT GGGGTTAGAC AATGTCCCCG TCACTTGTGC 21813 CTCCACCTGG CACCAGGCTA TGATGTTATG GCATTGAGGG TATGAGAAGG ACCAGGGGTT 21873 TCCCAGAGTT ACGCCCAGGC GCACAGGCAA TTGTTTCCTA CATGTGTGGC TGGAATGGTT 21933 GGGTGAGCCT TTTCAGCTGC CTACAATAGG AACCCAGGGA TACTGGGCAT TGACCAAGGC 21993 ATATCTCATA CCCTTTTCTT ATCTTTCTGC AG CAA ATT GTG GCT GTA AAT GTT 22046 Gln Ile Val Ala Val Asn Val 25 CCT CCT GAA GAT CAG GAT GGC TCT GGG GAT GAC TCT GAC AAC TTC TCT 22094 Pro Pro Glu Asp Gln Asp Gly Ser Gly Asp Asp Ser Asp Asn Phe Ser 30 35 40 45 GGC TCT GGC ACA GGTAAGACTG ACCCAGAACA CTGAGATGGC ATAGATCATG 22146 Gly Ser Gly Thr GCTGGAGTGG TGAGCAGGCA GTCACCCAGC TTTTAGTGAA CCCCCTTCTT CTCCCATCCC 22206 ATCCTTAGCC ATTGGAGTCA GGACAGTGCC AAAAGGAAGA ATGGTATCCA GCTGCAAGCC 22266 ACTCAGCTAA GAGAAACTCT CAGAGAAATG AAGGGGTTCC ACCAGGCCAT GGGCAGCCAC 22326 TAGAGCCAAC CCTTGGAGGA GTTTGACTCC ACTGAGCCTT GGTGTGGTGT TTCCATCTGT 22386 GAGATGGGAA TACTTTGCCC AAGAGCCTGT TAGAAGCTGT AGGAAGCACA GAGTCGGCTA 22446 GGTATAGATT TGCTCTCACC TCCATCTCTC GATACCAGTT CTCTGCAGAG CTTGGGTTTG 22506 TGGGAGGGGT GGGGGGGTGA GGGGAGAAGG CTGTGAGCTG CAGCTAGCCA GAGGGGTCTC 22566 CCAGAAGAAT GGGGAGAGCT AAGAAGGAAA GTTGAGGTCA CAGTGGGAAG GAGACCAGAG 22626 CAAAGGGTTG GAAGGTAGGT AAAATGCAGC CGTGTATTCT TGGGAGCCTT AGGCCTTGGG 22686 CAAGAGGGTA GAAGAGGTGT TTGTCCTGGG CTGCAGTCCT GTATCAGCTC TGGTGTCTTG 22746 GCCCACGCTC ACAGCAGGAT CCCTTCCCAG ATTCCCGAGA ATTTCTCACA GTTCAGAGAG 22806 CACGCTACTT GTAGGCAGGT GAGGCTGCAA AGGACAGCTT TTCTGGCCTA ATTTTCAAAG 22866 TGAGTTCAGC CTTTGCTAGG TCACCTTTGG GGTCTCAGAA GGCTTCAGCT CCTGGTAGAG 22926 CATGAATCAC GTCAGGCGTG ATGCTGGAGA CCTCTCCTAC CCTGACACCC CAAACCCCCA 22986 CCTCTGACCC TGCA GGT GCT TTG CCA GAT ACT TTG TCA CGG CAG ACA CCT 23036 Gly Ala Leu Pro Asp Thr Leu Ser Arg Gln Thr Pro 50 55 60 TCC ACT TGG AAG GAC GTG TGG CTG TTG ACA GCC ACG CCC ACA GCT CCA 23084 Ser Thr Trp Lys Asp Val Trp Leu Leu Thr Ala Thr Pro Thr Ala Pro 65 70 75 GAG CCC ACC AGC AGC AAC ACC GAG ACT GCT TTT ACC TCT GTC CTG CCA 23132 Glu Pro Thr Ser Ser Asn Thr Glu Thr Ala Phe Thr Ser Val Leu Pro 80 85 90 GCC GGA GAG AAG CCC GAG GAG GGA GAG CCT GTG CTC CAT GTA GAA GCA 23180 Ala Gly Glu Lys Pro Glu Glu Gly Glu Pro Val Leu His Val Glu Ala 95 100 105 GAG CCT GGC TTC ACT GCT CGG GAC AAG GAA AAG GAG GTC ACC ACC AGG 23228 Glu Pro Gly Phe Thr Ala Arg Asp Lys Glu Lys Glu Val Thr Thr Arg 110 115 120 125 CCC AGG GAG ACC GTG CAG CTC CCC ATC ACC CAA CGG GCC TCA ACA GTC 23276 Pro Arg Glu Thr Val Gln Leu Pro Ile Thr Gln Arg Ala Ser Thr Val 130 135 140 AGA GTC ACC ACA GCC CAG GCA GCT GTC ACA TCT CAT CCG CAC GGG GGC 23324 Arg Val Thr Thr Ala Gln Ala Ala Val Thr Ser His Pro His Gly Gly 145 150 155 ATG CAA CCT GGC CTC CAT GAG ACC TCG GCT CCC ACA GCA CCT GGT CAA 23372 Met Gln Pro Gly Leu His Glu Thr Ser Ala Pro Thr Ala Pro Gly Gln 160 165 170 CCT GAC CAT CAG CCT CCA CGT GTG GAG GGT GGC GGC ACT TCT GTC ATC 23420 Pro Asp His Gln Pro Pro Arg Val Glu Gly Gly Gly Thr Ser Val Ile 175 180 185 AAA GAG GTT GTC GAG GAT GGA ACT GCC AAT CAG CTT CCC GCA GGA GAG 23468 Lys Glu Val Val Glu Asp Gly Thr Ala Asn Gln Leu Pro Ala Gly Glu 190 195 200 205 GGC TCT GGA GAA CAA GTGAGTGGCT TTGCATTTCC TGGGTGGCCA CTAGTGCCTG 23523 Gly Ser Gly Glu Gln 210 CACCTGGCCG CCTAATGTCC TCATTACAGT GACAGGTGAC AGGGTCCCAC CTTCCTCCTG 23583 CCCGAAACAG ACTGATTGCA AGATCAGGAG GTGGGCGACT CCTTAGATGT CATTCAGGAG 23643 CTTACAGCAG GGTGAATTTT CCGTCTTAGA CCTTCATGGG AATTTTCACA CAACAATGTG 23703 TACGTTGTGT CACTGGAGGC GGTATCTGTG TCTTGGCCTG CCAGGGTCCC AGGTGTGACT 23763 GACTGCATTC CTTGACAGAT GCTGGTATAG GTTGGCTACG TCTGATGGGG GTGGCAGGGG 23823 ATCCCATCAG GTATGGCACT GCTCAGGTTG CTGTTGTGTC AGTGGCTCCA GCTGACCTGA 23883 TCCCAACCTA CCCTTCTGTA G GAC TTC ACC TTT GAA ACA TCT GGG GAG AAC 23934 Asp Phe Thr Phe Glu Thr Ser Gly Glu Asn 215 220 ACA GCT GTG GCT GCC GTA GAG CCC GGC CTG CGG AAT CAG CCC CCG GTG 23982 Thr Ala Val Ala Ala Val Glu Pro Gly Leu Arg Asn Gln Pro Pro Val 225 230 235 GAC GAA GGA GCC ACA GGT GCT TCT CAG AGC CTT TTG GAC AGG AAG GAA 24030 Asp Glu Gly Ala Thr Gly Ala Ser Gln Ser Leu Leu Asp Arg Lys Glu 240 245 250 GTG CTG GGA GGTGAGTCTT CTTTCAGGTG GAGAGGAGGA GGCAGGTGGT 24079 Val Leu Gly 255 GGCTCTGAGG TAGCCTGGGT TGCTGGGGTG AAGCATCTTT AGCAGCAGGG TGGGGAAGGA 24139 GGAGGGTCAA TTCTACTCCA GGCCACCTCC TAGGCTGTCC GTCTAGTCTG GGAGAGACTA 24199 CCACTGACCC CGTGGAGCTA CTGATCTGAG CCTGCCTCTG TTCACTCCCT A GGT GTC 24256 Gly Val ATT GCC GGA GGC CTA GTG GGC CTC ATC TTT GCT GTG TGC CTG GTG GCT 24304 Ile Ala Gly Gly Leu Val Gly Leu Ile Phe Ala Val Cys Leu Val Ala 260 265 270 TTC ATG CTG TAC CGG ATG AAG AAG AAG GAC GAA GGC AGC TAC TCC TTG 24352 Phe Met Leu Tyr Arg Met Lys Lys Lys Asp Glu Gly Ser Tyr Ser Leu 275 280 285 GAG GAG CCC AAA CAA GCC AAT GGC GGT GCC TAC CAG AAA CCC ACC AAG 24400 Glu Glu Pro Lys Gln Ala Asn Gly Gly Ala Tyr Gln Lys Pro Thr Lys 290 295 300 305 CAG GAG GAG TTC TAC GCC TGATGGGGAA ATAGTTCTTT CTCCCCCCAC 24448 Gln Glu Glu Phe Tyr Ala 310 AGCCCCTGCC ACTCACTAGG CTCCCACTTG CCTCTTCTGT GAAAAACTTC AAGCCCTGGC 24508 CTCCCCACCA CTGGGTCATG TCCTCTGCAC CCAGGCCCTT CCAGCTGTTC CTGCCCGAGC 24568 GGTCCCAGGG TGTGCTGGGA ACTGATTCCC CTCCTTTGAC TTCTGCCTAG AAGCTTGGGT 24628 GCAAAGGGTT TCTTGCATCT GATCTTTCTA CCACAACCAC ACCTGTTGTC CACTCTTCTG 24688 ACTTGGTTTC TCCAAATGGG AGGAGACCCA GCTCTGGACA GAAAGGGGAC CCGACTCTTT 24748 GGACCTAGAT GGCCTATTGC GGCTGGAGGA TCCTGAGGAC AGGAGAGGGG CTTCGGCTGA 24808 CCAGCCATAG CACTTACCCA TAGAGACCGC TAGGTTGGCC GTGCTGTGGT GGGGGATGGA 24868 GGCCTGAGCT CCTTGGAATC CACTTTTCAT TGTGGGGAGG TCTACTTTAG ACAACTTGGT 24928 TTTGCACATA TTTTCTCTAA TTTCTCTGTT CAGAGCCCCA GCAGACCTTA TTACTGGGGT 24988 AAGGCAAGTC TGTTGACTGG TGTCCCTCAC CTCGCTTCCC TAATCTACAT TCAGGAGACC 25048 GAATCGGGGG TTAATAAGAC TTTTTTTGTT TTTTGTTTTT GTTTTTAACC TAGAAGAACC 25108 AAATCTGGAC GGCAAAACGT AGGCTTAGTT TGTGTGTTGT CTCTGAGTTT GTCGCTCATG 25168 CGTACAACAG GGTATGGACT ATCTGTATGG TGCCCCATTT TTGGCGGCCC GTAAGTAGGC 25228 TGGCTAGTCC AGGATACTGT GGAATAGCCA CCTCTTGACC AGTCATGCCT GTGTGCATGG 25288 ACTCAGGGCC ACGGCCTTGG CCTGGGCCAC CGTGACATTG GAAGAGCCTG TGTGAGAACT 25348 TACTCGAAGT TCACAGTCTA GGAGTGGAGG GGAGGAGACT GTAGAGTTTT GGGGGAGGGG 25408 TGGCAAGGGT GCCCAAGCGT CTCCCACCTT TGGTACCATC TCTAGTCATC CTTCCTCCCG 25468 GAAGTTGACA AGACACATCT TGAGTATGGC TGGCACTGGT TCCTCCATCA AGAACCAAGT 25528 TCACCTTCAG CTCCTGTGGC CCCGCCCCCA GGCTGGAGTC AGAAATGTTT CCCAAAGAGT 25588 GAGTCTTTTG CTTTTGGCAA AACGCTACTT AATCCAATGG GTTCTGTACA GTAGATTTTG 25648 CAGATGTAAT AAACTTTAAT ATAAAGGAGT CCTATGAACT CTACTGCTTC TGCTTCTTCT 25708 TCTCTGGACT GGTGGTATAG ATATAGCCAC CCTTTGCCCA AACCCTGGTA GCTCGGGGAA 25768 GCTTGGCTTA AGGCTGCACG CCTCCAATCC CCCAAAGGTA GGATCCTGGC TGGGTCCAGG 25828 GTTCCTCTGA TTTATTTGGT TTTGTTGTGT TGTGTTGTGT TTTTCTTTTG GCTAAACTTC 25888 TTTTGGAAGT TGGTAAGTTC AGCCAAGGTT TTACAGGCCC TGATGTCTGT TCTTCTAAAT 25948 GGTTTAAGTA ATTGGGACTC TAGCACATCT TGACCTAGGG TCACTAGAGC TAAGCTTGCT 26008 TTGCAGGGCA GACACCTGGG ACAGCCTTCC TCCCTCATGT TTGCTGGGAC ACTGCTGAGC 26068 ACCCCTTGCT TACTTAGCTC AGTGATGTTC CAGCTCCTGG CTAGGCTGCT CAGCCACTCA 26128 GCTAGACAAA AGATCTGTGC CCTGTGTTTC ATCCCAGAGC TTGTTGCCAG ATCACATGGC 26188 TGGATGTGAT GTGGGGTGGG GGTGGGGTCA TATCTGAGAC AGCCCTCAGC TGAGGGCTTG 26248 TGGGACAGTG TCAAGCCTCA GGCTGGCGCT CATTCATATA ATTGCAATAA ATGGTACGTG 26308 TCCATTTGGA CAGCAGACAC TTTGGTGTAC TTGTGCAGTC TCTTTTTGGT CTGGACCATG 26368 TCCAACTCTA TCTGGTTTTT GGAATGGGAG CCTAACTGGC CTGTGTTCTG GCTTGGTACC 26428 AAATAGCAAC AGTCAGTGGC ATCCTTGCCC AGGCCCCAGG GCAGGACTAT GCTCTTGCCA 26488 TATCCAGGAC TCCCGACTTT GCACCTGTTT TCCCTCTGTG TGTAGCATCA TGAACTCCAG 26548 CTAGGTTGTT CCTTTCCCTG GGGTCAGGAG GATTCTGCTG ACTCTGAATG TCAGGATTTG 26608 CTTTTGTTCT GTTTGCTTAT TGGGCAATTC TCAACCTTCA CTAGCAACAG TCTCATGTGT 26668 CAGGATTACA AGTATTGCTT GCACATTGAG GG 26700 311 amino acids amino acid linear protein 2 Met Arg Arg Ala Ala Leu Trp Leu Trp Leu Cys Ala Leu Ala Leu Arg 1 5 10 15 Leu Gln Pro Ala Leu Pro Gln Ile Val Ala Val Asn Val Pro Pro Glu 20 25 30 Asp Gln Asp Gly Ser Gly Asp Asp Ser Asp Asn Phe Ser Gly Ser Gly 35 40 45 Thr Gly Ala Leu Pro Asp Thr Leu Ser Arg Gln Thr Pro Ser Thr Trp 50 55 60 Lys Asp Val Trp Leu Leu Thr Ala Thr Pro Thr Ala Pro Glu Pro Thr 65 70 75 80 Ser Ser Asn Thr Glu Thr Ala Phe Thr Ser Val Leu Pro Ala Gly Glu 85 90 95 Lys Pro Glu Glu Gly Glu Pro Val Leu His Val Glu Ala Glu Pro Gly 100 105 110 Phe Thr Ala Arg Asp Lys Glu Lys Glu Val Thr Thr Arg Pro Arg Glu 115 120 125 Thr Val Gln Leu Pro Ile Thr Gln Arg Ala Ser Thr Val Arg Val Thr 130 135 140 Thr Ala Gln Ala Ala Val Thr Ser His Pro His Gly Gly Met Gln Pro 145 150 155 160 Gly Leu His Glu Thr Ser Ala Pro Thr Ala Pro Gly Gln Pro Asp His 165 170 175 Gln Pro Pro Arg Val Glu Gly Gly Gly Thr Ser Val Ile Lys Glu Val 180 185 190 Val Glu Asp Gly Thr Ala Asn Gln Leu Pro Ala Gly Glu Gly Ser Gly 195 200 205 Glu Gln Asp Phe Thr Phe Glu Thr Ser Gly Glu Asn Thr Ala Val Ala 210 215 220 Ala Val Glu Pro Gly Leu Arg Asn Gln Pro Pro Val Asp Glu Gly Ala 225 230 235 240 Thr Gly Ala Ser Gln Ser Leu Leu Asp Arg Lys Glu Val Leu Gly Gly 245 250 255 Val Ile Ala Gly Gly Leu Val Gly Leu Ile Phe Ala Val Cys Leu Val 260 265 270 Ala Phe Met Leu Tyr Arg Met Lys Lys Lys Asp Glu Gly Ser Tyr Ser 275 280 285 Leu Glu Glu Pro Lys Gln Ala Asn Gly Gly Ala Tyr Gln Lys Pro Thr 290 295 300 Lys Gln Glu Glu Phe Tyr Ala 305 310 2196 base pairs nucleic acid double linear DNA (genomic) 3 TCTAGAACAC TTATTAAGAG CCAGGCACTG AAAAGTGCAG ACTCCCTCAT TTCATCCTGG 60 CCGTGCTTAC AAGTAGTTTC CATGCTCTGG TAACCCTGTG CAGAGGGCAG CGTGGGAGGC 120 GGGCCGCTTG GTGGACGGTC ATGGGGGCTC TGCATGGGTG GTTGCCCTTG CCTCAGAAGA 180 ACTCCCTAAG TAAGAGCAAG TTAGCCTCCC TAACCCCTGG TGGGTTGTTG CTTCTTTTCT 240 CCTCTTGTTT CTGCCAAGAG AGGGTGGACC AAGAAGACCC CAGCCTACAG AACATGTGAT 300 CCAAATAAAC TTCTTTTTAG TATAAATGTC CTAGCCTGTG ACGTTCTGGT AGACTAGCAC 360 AAGATGGACC AAGACAACTC TCATCGAGAC TCTGAGGAAC GAACTGGCAT CACATGGGAA 420 CAGGAAATGA AGCTTAGAGA GAGGTTCTGT GGCTTGTCCA ACATGGCTGT AGTTTAAATC 480 CAGCTTGCCA CCAAAGCACA CACATTTCAC TGCTGTGCTG GGCCGGGCCT CAGATCCCAG 540 GGGCTCCGGA GCTAGAAGGA CACGTGTATC AGCCATGGCT TCAGTTTATT GCTGTATACT 600 CTGTGCTTCT GGCTCTCATG GAAAAGACAG ACATTGGGGT TCTTATAATC TCTCCCTCTC 660 CCCTCCCCAC ACTCTATCCC CAAAGGAGGC ACCACTTCTG CAGGTAAATG TTATCTTCAA 720 AGCGCTCACA TCGCAACCTT TGCCCACACC ATCTCATTAA AGGAATTGGC AGTGACTTTA 780 AGGTGAAAGA ACTCGGTGGC TACGTGTTAT ATAAATTTGC ATCTGGGTCT CAGAGCTGGA 840 AGGAAGGCAC TCCCATACAT GCAGTCTGTA CATGCAGTCG GATGATGGAC CAACAACACA 900 TTGTGATTTA TGCCCCTGCT GGTGAGCCCA GGAATCCCTG TAGCACTCTC TCTCAGCTCT 960 AGGGCCCTGC TTGTGTATGG AAAACGCTTA GTGTTTTATA GGTATTTTGT CAGAATACTT 1020 TAAGGAACTT GACCAAAGTT ACAGGGAGGT TAGACAGATT GTCATGGTAT ACTCACCTCT 1080 GTCTCTGACC CTCCTAACTG GGACCTCTTT AGTCTCCCTT GAGGCAGGGA GTGCCACATG 1140 CATGTGTCCA GGCACATGTC TCCTGGTTTA CCTCCCAACG CACCTCAAGT CCCCAAGGTA 1200 GGTAGGCACT TGTATTCTGT AATTCAGAGA GGCAAATCAA ACTGTTACAA TGTTTGCCCA 1260 AAGCTCCCCA AGCAAAGTGG CCCTAAGAGT GAGCAAAGAG ACTGCGTGCC TTCACTGCCT 1320 GTGTGAATCC CTGCAGATAG TCTCTCATCT TGGTGCCCTT CCCACAGAGG CTGGGGCGGC 1380 AGGAGGGAGC CTGGACAGCT CAGACACTGG GTCATTGATG ACTGTTGTGT GGGATACCTG 1440 CCGGGGCGCA GGAGTGAGCC ATGCCACCCC AGGAAGTGGT TCAGGGTGAC TCTTCTTGGC 1500 ACACCTGGGA GGATGTAGCT GGTGCTGGCA CACCCACCGT CACGAGAGCT TCCTGTCCAA 1560 ACCTTCAACA AAGGCGGCTT CTTGAGACAG GCTAGACTGA AGTCACCAGC CTTGGGTGGG 1620 GTCCACTATG TAACCTCAGT GCTCAGGAAC CCTTTCCCAT ACTGTCTGGA ACTATACTGT 1680 ATGTAGCTGG GTTTCCACGC ATGTGTGCCT GCACCCAGTC CATCTCATCT TCTATCTCCC 1740 TCCCCTTTCC CGCTTCCCCC CTCCCCACTC TCCATCTCAT CTTCCATCCC CACCTCTTCT 1800 GGTCCCTGCC CTGCTAAACT CAGGGTAGCT GCATTCCGCT GGCCTTCCCC ATGTTCCAGG 1860 CTTCAGTCCC TTCTCTGCAC CTGTCCTTTG TGAAGTGACC AGAGGATTTC TGATCCTGTC 1920 TCTGTCGCTC TGAAGGGTCA GGAGTTCCTC CTGCCTGGAC AAAGCCATCC TGACGCACAT 1980 AAATAAAACA AACATCAAAC TCTATTCAAC CCCCTGGAAC CCGTGTGTGT TACTTACAGG 2040 GCAAAAGAAT GGAGCAGGGG ATGGGTTGTG GGGGGGGGGG GTGGCATCTG GGTTGTCTAC 2100 AGTTGTGCAT TAAGTTGTAA TTAAGATGTG CATTTCTCCA AATAAGGGAA AATTATTCTG 2160 GATTATTTGA GTGAAGCTGA AAGGTGATCA TCTAGA 2196 350 base pairs nucleic acid double linear DNA (genomic) 4 AGGCACATGT CTCCTGGTTT ACCTCCCAAC GCACCTCAAG TCCCCAAGGT AGGTAGGCAC 60 TTGTATTCTG TAATTCAGAG AGGCAAATCA AACTGTTACA ATGTTTGCCC AAAGCTCCCC 120 AAGCAAAGTG GCCCTAAGAG TGAGCAAAGA GACTGCGTGC CTTCACTGCC TGTGTGAATC 180 CCTGCAGATA GTCTCTCATC TTGGTGCCCT TCCCACAGAG GCTGGGGCGG CAGGAGGGAG 240 CCTGGACAGC TCAGACACTG GGTCATTGAT GACTGTTGTG TGGGATACCT GCCGGGGCGC 300 AGGAGTGAGC CATGCCACCC CAGGAAGTGG TTCAGGGTGA CTCTTCTTGG 350 283 base pairs nucleic acid double linear cDNA 5 TGCAGATAGT CTCTCATCTT GGTGCCCTTC CCACAGAGGC TGGGGCGGCA GGAGGGAGCC 60 TGGACAGCTC AGACACTGGG TCATTGATGA CTGTTGTGTG GGATACCTGC CGGGGCGCAG 120 GAGTGAGCCA TGCCACCCCA GGAAGTGGTT CAGGGTGACT CTTCTTGGCA CACCTGGGAG 180 GATGTAGCTG GTGCTGGCAC ACCCACCGTC ACGAGAGCTT CCTGTCCAAA CCTTCAACAA 240 AGGCGGCTTC TTGAGACAGG CTAGACTGAA GTCACCAGCC TTG 283 30 base pairs nucleic acid double linear cDNA 6 GCTGGCACAC CCACCGTCAC GAGAGCTTCC 30 20 base pairs nucleic acid double linear cDNA 7 TTGGCACACC TGGGAGGATG 20 19 base pairs nucleic acid double linear cDNA 8 AGTGGTTCAG GGTGACTCT 19 19 base pairs nucleic acid double linear cDNA 9 AGGAGTGAGC CATGCCACC 19 18 base pairs nucleic acid double linear cDNA 10 CTGGGTCATT GATGACTG 18 25 base pairs nucleic acid double linear DNA 11 TGGCACACCC ACCGTCACGA GAGCT 25 15 base pairs nucleic acid double linear DNA 12 TGGCACACCT GGGAG 15 15 base pairs nucleic acid double linear DNA 13 TGGTTCAGGG TGACT 15 39 base pairs nucleic acid double linear DNA 14 CTGGGTCATT GATGACTGTT GTGTGGGATA CCTGCCGGG 39 

What is claimed is:
 1. A method for promoting cutaneous wound healing, said method comprising: (a) providing, by directly introducing to a cutaneous wound site, a recombinant expression vector comprising the syndecan enhancer element, FiRE, operably linked to a FiRE-activated promoter wherein said syndecan enhancer element is less than or about 280 nucleotides of SEQ ID No. 5 in length and comprises binding sites for nuclear proteins FIN-1, AP-1 and USF, and wherein said FiRE activated promoter is operably linked to a structural gene encoding a growth factor that promotes said cutaneous wound healing; and (b) inducing expression of said structural gene encoding said growth factor by activating said syndecan enhancer element, wherein expression of said growth factor promotes said cutaneous wound healing.
 2. The method of claim 1, wherein said FiRE activated promoter is a syndecan promoter.
 3. The method of claim 1, wherein said expression occurs in the edge of an incision site.
 4. The method of claim 1, wherein said expression occurs in keratinocytes.
 5. The method of claim 1, wherein said growth factor is FGF-7.
 6. The method of any one of claims 1, 2-4 and 5, wherein said expression of said growth factor that promotes said cutaneous wound healing promotes tissue regeneration.
 7. A method for promoting regeneration of tissue, said method comprising: (a) providing, by directly introducing to a tissue, a recombinant expression vector comprising the syndecan enhancer element, FiRE, operably linked to a FiRE-activated promoter wherein said syndecan enhancer element is less than or about 280 nucleotides of SEQ ID No. 5 in length and comprises binding sites for nuclear proteins FIN-1, AP-1 and USF, and wherein said FiRE activated promoter is operably linked to a structural gene encoding a growth factor that promotes said regeneration of said tissue; and (b) inducing expression of said structural gene encoding said growth factor by activating said syndecan enhancer element, wherein expression of said growth factor promotes said regeneration of said tissue.
 8. The method of claim 7, wherein said promoter is a syndecan promoter.
 9. The method of claim 7, wherein said expression occurs in the edge of an incision site.
 10. The method of claim 7, wherein said expression occurs in keratinocytes.
 11. The method of claim 7, wherein said growth factor is FGF-7. 